Elongase gene, and process for the preparation of polyunsaturated fatty acids

ABSTRACT

The invention relates to novel elongase genes with the sequences stated in sequence SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 or their homologs, derivatives or analogs, to a gene construct comprising this gene or its homologs, derivatives and analogs, and to its use. The invention also relates to vectors or transgenic organisms comprising an elongase gene with the sequence SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 or its homologs, derivatives and analogs. The invention furthermore relates to the use of the elongase gene sequences alone or in combination with further elongases and/or further fatty acid biosynthesis genes. The present invention relates to a novel elongase gene with the sequence SEQ ID NO:1 or its homologs, derivatives and analogs. Furthermore, the invention relates to a process for the preparation of polyunsaturated fatty acids and to a process for introducing DNA into organisms which produce large amounts of oils and, in particular, oils with a high content of unsaturated fatty acids. Moreover, the invention relates to an oil and/or a fatty acid preparation with a higher content of polyunsaturated fatty acids with at least two double bonds and/or a triacylglycerol preparation with a higher content of polyunsaturated fatty acids with at least two double bonds.

This is a Divisional application of application Ser. No. 10/182,634filed on Jul. 31, 2002 (now U.S. Pat. No. 7,544,859), the entiredisclosure of which is hereby incorporated by reference, which is anational stage entry of PCT/EP01/01346 filed on Feb. 8, 2001.

FIELD OF THE INVENTION

The invention relates to a novel elongase gene with the sequences statedin sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9 and SEQ ID NO:11 or their homologs, derivatives or analogs, to agene construct comprising this gene or its homologs, derivatives andanalogs, and to its use. The invention also relates to vectors ortransgenic organisms comprising an elongase gene with the sequence SEQID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ IDNO:11, or its homologs, derivatives and analogs. The inventionfurthermore relates to the use of the elongase gene sequences alone orin combination with further elongases and/or further fatty acidbiosynthesis genes. The present invention relates to a novel elongasegene with the sequence SEQ ID NO:1 or its homologs, derivatives andanalogs.

Furthermore, the invention relates to a process for the preparation ofpolyunsaturated fatty acids and to a process for introducing DNA intoorganisms which produce large amounts of oils and, in particular, oilswith a high content of unsaturated fatty acids. Moreover, the inventionrelates to an oil and/or a fatty acid preparation with a higher contentof polyunsaturated fatty acids with at least two double bonds and/or atriacylglycerol preparation with a higher content of polyunsaturatedfatty acids with at least two double bonds.

BACKGROUND OF THE INVENTION

Certain products and byproducts of naturally occurring metabolicprocesses in cells can be used for a wide spectrum of industries,including the animal feed industry, food industry, cosmetics industryand pharmaceuticals industry. These molecules, which are joinly referredto as “fine chemicals”, also include lipids and fatty acids, amongstwhich the polyunsaturated fatty acids constitute an example of oneclass. Polyunsaturated fatty acids (PUFAs) are added, for example, tochildren's formula to increase its nutritional value. For example, PUFAshave a positive effect on the cholesterol level in the blood of humansand are therefore suitable for protection against heart disease. Finechemicals and polyunsaturated fatty acids (PUFAs) can be isolated fromanimal sources, for example fish, or microorganisms. Culturing thesemicroorganisms allows large amounts of one or more of the desiredmolecules to be produced and isolated.

Microorganisms which are especially suitable for preparing PUFAs aremicroorganisms such as Thraustochytria or Schizochytria strains, algaesuch as Phaeodactylum tricornutum or Crypthecodinium species, Ciliatasuch as Stylonychia or Colpidium, fungi such as Mortierella,Entomophthora or Mucor. A number of mutant strains of the microorganismsin question which produce a series of desirable compounds, includingPUFAs, have been developed by strain selection. The selection of strainswith an improved production of a certain molecule is, however, a timeconsuming and difficult procedure. Also disadvantageous is the fact thatonly specific unsaturated fatty acids, or only a specific fatty acidspectrum, can be produced by a defined microorganism.

As an alternative, fine chemicals can suitably be produced on a largescale via the production of plants which have been developed in such away that they produce the abovementioned PUFAs. Plants which areparticularly well suited to this purpose are oil crops which containlarge amounts of lipid compounds, such as oilseed rape, canola, linseed,soya, sunflowers, borage and evening primrose. However, other cropswhich contain oils or lipids and fatty acids are well suited, asmentioned in the detailed description of the present invention.Conventional plant breeding has led to the development of a series ofmutant plants which produce a spectrum of desirable lipids and fattyacids, cofactors and enzymes. However, the selection of novel plantvarieties with an improved production of a certain molecule is atime-consuming and difficult procedure or even impossible if thecompound does not occur naturally in the plant in question, such as inthe case of polyunsaturated C₂₀-fatty acids, and C₂₂-fatty acids andthose with longer carbon chains.

The invention provides novel nucleic acid molecules which are suitablefor identifying and isolating elongase genes of PUFA biosynthesis andwhich can be used for the modification of oils, fatty acids, lipids,lipid-derived compounds and, most preferably, for the preparation ofpolyunsaturated fatty acids, since there remains a great demand fornovel genes which encode enzymes which are involved in the biosynthesisof unsaturated fatty acids and which make it possible for these to beprepared on an industrial scale. In particular, there is a demand forfatty acid biosynthesis enzymes which make possible the elongation ofpolyunsaturated fatty acids, preferably with two or more double bonds inthe molecule. The nucleic acids according to the invention encodeenzymes which have this ability.

Microorganisms such as Phaeodactylum, Colpidium, Mortierella,Entomophthora, Mucor, Crypthecodinium and other algae and fungi andplants, in particular oil crops, are generally used in industry for theproduction of a large number of fine chemicals on a large scale.

As long as cloning vectors and techniques are available for the geneticmanipulation of the abovementioned microorganisms and Ciliata, asdisclosed in WO 98/01572 and WO 00/23604, or algae and relatedorganisms, such as Phaeodactylum tricornutum, described in Falciatore etal. [1999, Marine Biotechnology 1(3):239-251]; and Dunahay et al. [1995,Genetic transformation of diatoms, J. Phycol. 31:10004-1012] and thereferences cited therein, the nucleic acid molecules according to theinvention can be used for the recombinant modification of theseorganisms so that they become better or more efficient producers of oneor more fine chemicals, especially unsaturated fatty acids. Thisimproved production or production efficiency of a fine chemical can becaused by a direct effect of manipulating a gene according to theinvention or by an indirect effect of this manipulation.

Mosses and algae are the only known plant systems which produceconsiderable amounts of polyunsaturated fatty acids, such as arachidonicacid (=ARA) and/or eicosapentaenoic acid (=EPA) and/or docosahexaenoicacid (=DHA). Mosses contain PUFAs in membrane lipids, while algae,organisms related to algae and some fungi also accumulate considerableamounts of PUFAs in the triacylglycerol fraction. Thus, nucleic acidmolecules which are isolated from such strains which also accumulatePUFAs in the triacylglycerol fractions are particularly suitable formodifying the lipid and PUFA production systems in a host, in particularin microorganisms, such as the abovementioned microorganisms, andplants, such as oil crops, for example oilseed rape, canola, linseed,soya, sunflower, borage, castor-oil plant, oil palm, safflower(Carthamus tinctorius), coconut, peanut or cacao bean. Furthermore,nucleic acids from triacylglycerol-accumulating microorganisms can beused for identifying such DNA sequences and enzymes in other specieswhich are suitable for modifying the biosynthesis of PUFA precursormolecules in the organisms in question. Microorganisms which accumulatePUFAs such as ARA, EPA or DHA in triacylglycerols are, in particular,microorganisms such as Crypthecodinium cohnii and Thraustochytriumspecies. Thraustochytria are also closely related to the Schizochytriastrains in terms of phylogenetics. Even though these organisms are notclosely related to mosses such as Physcomitrella, sequence similaritiesat the DNA sequence and, in particular, polypeptide level can beobserved to such an extent that DNA molecules can be identified,isolated and characterized functionally in heterologous hybridizationexperiments, sequence alignments and experiments using the polymerasechain reaction, even from organisms which are distantly related in termsof evolution. In particular, consense sequences can be derived which aresuitable for the heterologous screening or the functionalcomplementation and prediction of gene functions in third species. Theability to identify these functions, for example to predict thesubstrate specificity of enzymes, can therefore be of significantimportance. Furthermore, these nucleic acid molecules may act asreference sequences for mapping related genomes or for deriving PCRprimers.

The novel nucleic acid molecules encode proteins termed in the presentcontext PUFA-specific elongases (=PSEs, or PSE in the singular). ThesePSEs can, for example, exert a function which is involved in themetabolism (for example in the biosynthesis or in the breakdown) ofcompounds required for lipid or fatty acid synthesis, such as PUFAs, orwhich participate in the transmembrane transport of one or morelipid/fatty acid compositions, either into the cell or out of the cell.

This novel application shows the isolation of such novel elongase genesin greater detail. For the first time, we have isolated elongase geneswhich are suitable for producing long-chain polyunsaturated fatty acids,preferably having more than eighteen or twenty carbon atoms in thecarbon skeleton of the fatty acid and/or at least two double bonds inthe carbon chain while being derived from typical organisms whichcontain high amounts of PUFAs in the triacylglycerol fraction. Thismeans, in the singular, a PSE gene or PSE protein or, in the plural, PSEgenes or PSE proteins. Other known patent applications and publicationsdisclose, or show, no functionally active PSE gene, even though variousknown patent applications exist which show the elongation of saturatedfatty acids of short or medium chain length (WO 98/46776 and U.S. Pat.No. 5,475,099) or the elongation or production of long-chain fattyacids, but which then have no more than one double bond or lead tolong-chain fatty acid wax esters (see WO 98/54954, WO 96/13582, WO95/15387). The invention presented here describes the isolation of novelelongases with novel properties. Starting from the sequence stated inSEQ ID NO:1, it was possible to find further nucleic acids which encodeelongases which elongate unsaturated fatty acids.

WO 99/64616, WO 98/46763, WO 98/46764 and WO 98/46765 describe theproduction of PUFAs in transgenic plants and demonstrate the cloning andfunctional expression of corresponding desaturase activities, inparticular from fungi, but demonstrate no PSE-encoding gene and nofunctional PSE activity. The expression of the desaturase activitiesleads to a shift in the fatty acid spectrum in the transgenic plants,but no increased content of unsaturated fatty acids was observed. Theproduction of a trienoic acid with C₁₈-carbon chain has beendemonstrated and claimed with reference to gamma-linolenic acid, but theproduction of very long-chain polyunsaturated fatty acids (with a C₂₀—and longer carbon chain and of trienoic acids and higher unsaturatedtypes) has, however, not been demonstrated to date.

To prepare long-chain PUFAs, the polyunsaturated C₁₆- or C₁₈-fatty acidsmust be elongated by at least two carbon atoms by the enzymatic activityof an elongase. The nucleic acid sequence SEQ ID NO:1 according to theinvention enclodes the first plant elongase which is capable ofelongating the C₁₆- or C₁₈-fatty acids with at least two double bonds inthe fatty acid by at least two carbon atoms. After one elongation cycle,this enzyme activity leads to C₂₀-fatty acids, and after two, three andfour elongation cycles to C₂₂-, C₂₄- or C₂₆-fatty acids. Longer-chainPUFAs can also be synthesized with the aid of the other elongases whichare disclosed (SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 andSEQ ID NO:11). They can be employed individually, multiply or, forexample, in addition to the PUFA elongase from the moss Physcomitrellapatens (SEQ ID NO:1) for increasing the PUFA content in a novel processfor the preparation of PUFAs. The activity of the elongases according tothe invention preferably leads to C₂₀-fatty acids with at least twodouble bonds in the fatty acid molecule, preferably with three or fourdouble bonds, especially preferably three double bonds, in the fattyacid molecule and/or C₂₂-fatty acids with at least two double bonds inthe fatty acid molecule, preferably with four, five or six double bonds,especially preferably with five or six double bonds, in the molecule.After the elongation by the enzyme according to the invention has takenplace, further desaturation steps may be carried out in order to obtainthe highly desaturated fatty acids. The products of the elongaseactivities and of the further desaturation with is a possibilitytherefore lead to preferred PUFAs with a higher degree of desaturation,such as docosadienoic acid, arachidonic acid,ω6-eicosatrienedihomo-γ-linolenic acid, eicosapentaenoic acid,ω3-eicosatrienoic acid, ω3-eicosatetraenoic acid, docosapentaenoic acidor docosahexaenoic acid. Substrates of the enzyme activity according tothe invention are, for example, taxol acid; 7,10,13-hexadecatrienoicacid, 6,9-octadecadienoic acid, linolic acid, linolenic acid, α- orγ-linolenic acid or stearidonic acid, and also arachidonic acid,eicosatetraenoic acid, docosapentaenoic acid, eicosapentaenoic acid.Preferred substrates are linolic acid, γ-linolenic acid and/orα-linolenic acid, and also arachidonic acid, eicosatetraenoic acid,docosapentaenoic acid and eicosapentaenoic acid. Arachidonic acid,docosapentaenoic acid and eicosapentaenoic acid are especiallypreferred. The C₁₆- or C₁₈-fatty acids with at least two double bonds inthe fatty acid can be elongated by the enzymatic activity according tothe invention in the form of the free fatty acid or in the form of theesters, such as phospholipids, glycolipids, sphingolipids,phosphoglycerides, monoacylglycerol, diacylglycerol or triacylglycerol.

Of particular importance for human nutrition is conjugated linolic acid“CLA”. CLA is to be understood as meaning, in particular, fatty acidssuch as C18:2^(9 cis, 11trans) or the isomer C18:2^(10trans, 12 cis),which can be desaturated or elongated after uptake in the body owing tohuman enzyme systems and can contribute to health-promoting effects.Elongases according to the invention also allow those conjugated fattyacids which have at least two double bonds in the molecule to beelongated and thus make available such health-promoting fatty acids forhuman nutrition. Other examples of conjugated fatty acids arealpha-parinaric acid, eleostearic acid and calendulic acid.

Given cloning vectors for use in plants and in the transformation ofplants, such as those which are published, and cited, in: PlantMolecular Biology and Biotechnology (CRC Press, Boca Raton, Fla.),chapter 6/7, pp. 71-119 (1993); F. F. White, Vectors for Gene Transferin Higher Plants; in: Transgenic Plants, Vol. 1, Engineering andUtilization, Ed.: Kung and R. Wu, Academic Press, 1993, 15-38; B. Jeneset al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1,Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press (1993),128-143; Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42(1991), 205-225)), the nucleic acids according to the invention can beused for the recombinant modification of a broad spectrum of plants sothat they become a better, more efficient or modified producer of one ormore lipid-derived products, such as PUFAs. This improved production orproduction efficiency of a lipid-derived product, such as PUFAs, can becaused by the direct effect of the manipulation or by an indirect effectof this manipulation.

There exists a series of mechanisms by which the modification of a PSEprotein according to the invention can directly affect yield, productionand/or production efficiency of a fine chemical from an oil crop or amicroorganism, owing to a modified protein. The number or activity ofthe PSE protein or PSE gene can be increased so that greater quantitiesof these compounds are produced de novo since the organisms lacked thisactivity and biosynthesis ability prior to introduction of the gene inquestion. Also, the use of various, divergent sequences, i.e. sequenceswhich differ at the DNA sequence level, may be advantageous in thiscontext.

The introduction of a PSE gene or a plurality of PSE genes to anorganism or a cell can not only increase the biosynthesis flow towardthe end product, but also increase, or create de novo, the correspondingtriacylglycerol composition. Equally, the number or activity of othergenes which are involved in the import of nutrients required for thebiosynthesis of one or more fine chemicals (for example fatty acids,polar and neutral lipids) may be increased, so that the concentration ofthese precursors, cofactors or intermediates is increased within thecells or within the storage compartment, thus further increasing theability of the cells to produce PUFAs, as described hereinbelow. Fattyacids and lipids themselves are desirable as fine chemicals;optimization of the activity, or increasing the number, of one or morePSEs which are involved in the biosynthesis of these compounds, ordestroying the activity of one or more PSEs which are involved in thebreakdown of these compounds, can make possible an increase in yield,production and/or production efficiency of fatty acid molecules andlipid molecules from plants or microorgansims.

The mutagenesis of the PSE gene according to the invention may also leadto a PSE protein with modified activities which directly or indirectlyaffect the production of one or more desired fine chemicals. Forexample, the number or activity of the PSE gene according to theinvention can be increased, so that the normal metabolic waste productsor byproducts of the cell (whose quantity might be increased owing tothe overproduction of the desired fine chemical) are exported in anefficient manner before they destroy other molecules or processes withinthe cell (which would reduce cell viability) or would interfere with thebiosynthetic pathways of the fine chemical (thus reducing yield,production or production efficiency of the desired fine chemical).Furthermore, the relatively large intracellular quantities of thedesired fine chemical themselves may be toxic to the cell or mayinterfere with enzyme feedback mechanisms, such as allostericregulation; for example, they might increase the allocation of the PUFAinto the triacylglycerol fraction owing to an increased activity ornumber of other enzymes or detoxifying enzymes of the PUFA pathway whichfollow downstream; the viability of the seed cells might increase which,in turn, leads to better development of cells in culture or to seedswhich produce the desired fine chemical. Alternatively, the PSE geneaccording to the invention can be manipulated in such a way that thecorresponding quantities of the various lipid molecules and fatty acidmolecules are produced. This can have a decisive effect on the lipidcomposition of the cell membrane and generates novel oils in addition tothe occurrence of PUFAs which have been synthesized de novo. Since eachtype of lipid has different physical properties, a change in the lipidcomposition of a membrane can substantially modify membrane fluidity.Changes in membrane fluidity can have an effect on the transport ofmolecules via the membrane and on cell integrity, both of which have adecisive effect on the production of fine chemicals. In plants,moreover, these changes can also have an effect on other traits such asthe tolerance to abiotic and biotic stress situations.

Biotic and abiotic stress tolerance is a general trait which it isdesirable to impart to a broad spectrum of plants such as maize, wheat,rye, oats, triticale, rice, barley, soybean, peanut, cotton, oilseedrape and canola, cassava, pepper, sunflower and tagetes, Solanaceaeplants such as potato, tobacco, aubergine and tomato, Vicia species,pea, alfalfa, shrub plants (coffee, cacao, tea), Salix species, trees(oil palm, coconut) and perennial grasses and fodder crops. As a furtherembodiment according to the invention, these crops are also preferredtarget plants for genetic engineering. Very especially preferred plantsaccording to the invention are oil crops such as soybean, peanut,oilseed rape, canola, sunflower, safflower, trees (oil palm, coconut) orcrops such as maize, wheat, rye, oats, triticale, rice, barley, alfalfa,or shrub plants (coffee, cacao, tea).

Accordingly, one aspect of the invention relates to isolated nucleicacid molecules (for example cDNAs), encompassing nucleotide sequenceswhich encode a PSE or several PSEs or biologically active parts thereof,or nucleic acid fragments which are suitable as primers or hybridizationprobes for the detection or amplification of PSE-encoding nucleic acids(for example DNA or mRNA). In a specially preferred embodiment, thenucleic acid molecule encompasses one of the nucleotide sequences shownin SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 andSEQ ID NO:11, or the coding region or a complement of one of thesenucleotide sequences. In other especially preferred embodiments, theisolated nucleic acid molecule according to the invention encompasses anucleotide sequence which hybridizes with a nucleotide sequence as shownin the sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQID NO:9 and SEQ ID NO:11, or a part thereof or which has at leastapproximately 50%, preferably at least approximately 60%, morepreferably at least approximately 70%, 80% or 90% and even morepreferably at least approximately 95%, 96%, 97%, 98%, 99% or morehomology thereto. In other preferred embodiments, the isolated nucleicacid molecule encodes one of the amino acid sequences shown in thesequence SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10 and SEQ ID NO:12. Preferably, the preferred PSE gene according tothe invention also has at least one of the PSE activities describedherein.

In a further embodiment, the isolated nucleic acid molecule encodes aprotein or part thereof, the protein or the part thereof comprising anamino acid sequence which has sufficiently homology with an amino acidsequence of the sequence SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10 and SEQ ID NO:12, that the protein or the partthereof retains a PSE activity. Preferably, the protein or the partthereof which is encoded by the nucleic acid molecule retains theability to participate in the metabolism of compounds required for thesynthesis of cell membranes of plants or in the transport of moleculesvia these membranes. In one embodiment, the protein encoded by thenucleic acid molecule has at least approximately 50%, preferably atleast approximately 60% and more preferably at least approximately 70%,80% or 90% and most preferably at least approximately 95%, 96%, 97%,98%, 99% or more homology with an amino acid sequence of the sequenceSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQID NO:12. In a further preferred embodiment, the protein is afull-length protein, parts of which are essentially homologous to acomplete amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12, (which is due to the openreading frame shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9 and SEQ ID NO:11) and which can be isolated in itsfull length by methods and experiments with which the skilled worker isfamiliar.

In another preferred embodiment, the isolated nucleic acid moleculeoriginates from Phytophthora infestans, Physcomitrella patens,Crypthecodinium cohnii or Thraustochytrium and encodes a protein (forexample a PSE fusion protein) comprising a biologically active domainwhich has at least approximately 50% or more homology with an amino acidsequence of the sequence SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10 and SEQ ID NO:12 and retains the ability toparticipate in the metabolism of compounds required for the synthesis ofcell membranes of plants or in the transport of molecules via thesemembranes or which has at least one of the elongation activitiesresulting in PUFAs such as ARA, EPA or DHA or their precursor moleculesor one of the activities listed in Table 1, and also encompassesheterologous nucleic acid sequences which encode a heterologouspolypeptide or regulatory proteins.

TABLE 1 Fatty acid profile of five transgenic yeast strains in mol %.The proportions of γ-linolenic acid which has been added and taken upare emphasized by numbers printed in bold, those of the elongatedproducts are underlined and those of the elongated γ-linolenic acid areemphasized by numbers printed in bold (last line). Fatty acids [mol %]pYES2 pY2PSE1a pY2PSE1b pY2PSE1c pY2PSE1d 16:0 17.0 17.6 16.4 16.3 17.616:1Δ⁹ 28.0 26.8 28.0 27.9 25.1 18:0 6.5 6.0 6.4 5.6 6.1 18:1Δ⁹ 25.923.5 27.0 25.2 21.4 18:3Δ^(6,9,12) 22.6 15.7 13.2 16.4 22.820:3Δ^(8,11,14) — 10.3 9.0 8.6 7.1 18:3Δ^(6,9,12) — 39.6 40.5 34.4 23.7Elongation

In another embodiment, the isolated nucleic acid molecule is at least 15nucleotides in length and hybridizes under stringent conditions with anucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11. Theisolated nucleic acid molecule preferably corresponds to a naturallyoccurring nucleic acid molecule. More preferably, the isolated nucleicacid molecule encodes naturally occurring Crypthecodinium, Phytophthoraor Thraustochytrium PSE or a biologically active part thereof.

A further aspect of the invention relates to vectors, for examplerecombinant expression vectors, comprising at least one nucleotidemolecule according to the invention and host cells into which thesevectors have been introduced, in particular microorganisms, plant cells,plant tissues, plant organs or intact plants. In one embodiment, such ahost cell can store fine chemicals, in particular PUFAs; to isolate thedesired compound, the cells are harvested. The compound (oils, lipids,triacylglycerides, fatty acids) or the PSE can then be isolated from themedium or from the host cell which, in the case of plants, are cellscomprising or storing the fine chemicals, most preferably cells ofstorage tissues such as seed coats, tubers, epidermis cells and seedcells.

Yet another aspect of the invention relates to a genetically modifiedplant, preferably an oil crop as mentioned above, especially preferablya rapeseed, linseed or Physcomitrella patens plant into which a PSE genehas been introduced. In one embodiment, the genome of oilseed rape,linseed or Physcomitrella patens has been modified by introducing, astransgene, a nucleic acid molecule according to the invention encoding awild-type or mutated PSE sequence. In another embodiment, an endogenousPSE gene in the genome of the donor organisms Physcomitrella patens,Phytophthora infestans, Crypthecodinium or Thraustochytrium has beenmodified, that is to say functionally destroyed, for example byhomologous recombination with a modified PSE gene or by mutagenesis anddetection by means of DNA sequences. In a preferred embodiment, theplant organism belongs to the genus Physcomitrella, Ceratodon, Funaria,oilseed rape or linseed, with Physcomitrella, oilseed rape or linseedbeing preferred. In a preferred embodiment, Physcomitrella, oilseed rapeor linseed is also used to produce a desired compound such as lipids orfatty acids, with PUFAs being especially preferred.

In yet another preferred embodiment, the moss Physcomitrella patens canbe used for demonstrating a function of an elongase gene usinghomologous recombination on the basis of the nucleic acids described inthe present invention.

Yet another aspect of the invention relates to an isolated PSE gene or apart, for example a biologically active part, thereof. In a preferredembodiment, the isolated PSE or a part thereof can participate in themetabolism of compounds required for the synthesis of cell membranes ina microorganism or a plant cell or in the transport of molecules via itsmembranes. In a further preferred embodiment, the isolated PSE or thepart thereof has sufficient homology with an amino acid sequence of SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ IDNO:12 for this protein or the part thereof to retain the ability toparticipate in the metabolism of compounds required for the synthesis ofcell membranes in microorganisms or plant cells or in the transport ofmolecules via these membranes.

The invention also provides an isolated preparation of a PSE. Inpreferred embodiments, the PSE gene encompasses an amino acid sequenceof SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 andSEQ ID NO:12. In a further preferred embodiment, the invention relatesto an isolated full-length protein which is essentially homologous witha complete amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12 (which are encoded by theopen reading frames shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQID NO:7, SEQ ID NO and SEQ ID NO:11). In a further embodiment, theprotein has at least approximately 50%, preferably at leastapproximately 60%, more preferably at least approximately 70%, 80% or90% and most preferably at least approximately 95%, 96%, 97%, 98%, 99%or more homology with an amino acid sequence of sequence SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12. Inother embodiments, the isolated PSE encompasses an amino acid sequencewhich has at least approximately 50% homology with one of the amino acidsequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10 and SEQ ID NO:12 and which can participate in the metabolism ofcompounds required for the synthesis of fatty acids in a microorganismor a plant cell or in the transport of molecules via these membranes orhas one or more of the PUFA-elongating activities, the elongationadvantageously concerning desaturated C₁₆- or C₁₈- or C₂₀-carbon chainswith double bonds in at least two positions.

As an alternative, the isolated PSE can encompass an amino acid sequencewhich is encoded by a nucleotide sequence hybridizing with a nucleotidesequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9 and SEQ ID NO:11, for example under stringent conditions, or whichhas at least approximately 50%, preferably at least approximately 60%,more preferably at least approximately 70%, 80% or 90% and even morepreferably at least approximately 95%, 96%, 97%, 98%, 99% or morehomology thereto. It is also preferred for the preferred PSE forms alsoto have one of the PSE activities described herein.

The PSE polypeptide or a biologically active part thereof can be linkedfunctionally to a non-PSE polypeptide to form a fusion protein. Inpreferred embodiments, this fusion protein has an activity which differsfrom that of PSE alone. In other preferred embodiments, this fusionprotein participates in the metabolism of compounds which are requiredfor the synthesis of lipids and fatty acids, cofactors and enzymes inmicroorganisms or plants, or in the transport of molecules via thesemembranes. In especially preferred embodiments, the introduction of thisfusion protein into a host cell modulates the production of a desiredcompound by the cell. In a preferred embodiment, these fusion proteinsalso contain Δ4-, Δ5- or Δ6-, Δ8-, Δ15-, Δ17- or Δ19-desaturaseactivities, alone or in combination.

Another aspect of the invention relates to a process for the productionof a fine chemical. This process either comprises culturing a suitablemicroorganism or culturing plant cells, plant tissues, plant organs orintact plants encompassing the nucleotide sequences according to theinvention of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9 and SEQ ID NO:11 or their homologs, derivatives or analogs or agene construct which compasses SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11 or their homologs, derivativesor analogs, or a vector encompassing these sequences or the geneconstruct which brings about the expression of a PSE nucleic acidmolecule according to the invention so that a fine chemical is produced.In a preferred embodiment, the process furthermore encompasses the stepof obtaining a cell comprising such an elongase nucleic acid sequenceaccording to the invention, in which a cell is transformed with anelongase nucleic acid sequence, a gene construct or a vector which bringabout the expression of a PSE nucleic acid according to the invention.In a further preferred embodiment, this process furthermore comprisesthe step of obtaining the fine chemical from the culture. In anespecially preferred embodiment, the cell belongs to the order of theCiliata, to microorganisms such as fungi, or to the plant kingdom, inparticular to oil crops, with microorganisms or oil crops beingespecially preferred.

A further aspect of the invention relates to methods of modulating theproduction of a molecule by a microorganism. These methods encompasscombining the cell with a substance which modulates the PSE activity orthe expression of the PSE nucleic acid so that a cell-associatedactivity is modified relative to the same activity in the absence of thesubstance. In a preferred embodiment, a metabolic pathway, or twometabolic pathways, of the cell for lipids and fatty acids, cofactorsand enzymes is, or are, modulated or the transport of compounds viathese membranes is modulated so that the yield or the production rate ofa desired fine chemical by this microorganism is improved. The substancewhich modulates the PSE activity can be a substance which stimulates thePSE activity or the expression of the PSE nucleic acid or which can beused as intermediate in fatty acid biosynthesis. Examples of substanceswhich stimulate the PSE activity or the expression of PSE nucleic acidsare, inter alia, small molecules, active PSEs and nucleic acids encodingPSEs which have been introduced into the cell. Examples of substanceswhich inhibit the PSE activity or PSE expression are, inter alia, smallmolecules and/or antisense PSE nucleic acid molecules.

A further aspect of the invention relates to methods of modulating theyields of a desired compound from a cell, which encompass introducing,into a cell, a wild-type or mutant PSE gene which is either kept on aseparate plasmid or integrated into the genome of the host cell. In thecase of integration into the genome, integration can be random or takeplace by recombination in such a way that the native gene is replaced bythe copy which is introduced, thus modulating the production of thedesired compound by the cell, or by using a gene in trans, so that thegene is functionally linked to a functional expression unit comprisingat least one sequence which ensures the expression of a gene and atleast one sequence which ensures the polyadenylation of a functionallytranscribed gene.

In a preferred embodiment, the yields are modified. In a furtherembodiment, the desired chemical is increased, it being possible toreduce undesired compounds which have a negative effect. In anespecially preferred embodiment, the desired fine chemical is a lipid orfatty acid, a cofactor or an enzyme. In an especially preferredembodiment, this chemical is a polyunsaturated fatty acid. Morepreferably, it is selected from amongst arachidonic acid (=ARA),eicosapentaenoic acid (=EPA) or docosahexaenoic acid (=DHA).

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary methods and arrangements conducted and configured according tothe advantageous solutions presented herein are depicted in theaccompanying drawings wherein:

FIG. 1: illustrates a sequence alognment of the yeast elol peptide(upper row) with physcomitrella patens PpSE1 (SEQ ID NO:2);

FIGS. 2 a-2 e: illustrate GC analyses of FAMEs which from total lipidsof yeast transformed with pYES2 (i/control) and pY2PSE1 (ii-iv-c+d) ineach case transformed with pY2PSE1A, pY2PSE1B, pY2PSE1C, and pY2PSE1D;

FIGS. 3 a-b: illustrate the structure and mass spectra of the DMOXderivatives of cis-Δ6, 9, 12, C18:3;

FIGS. 4 a-b: illustrate the structure and mass spectra of the DMOXderivatives of cis-Δ8, 11, 14, C20:3;

FIG. 5: illustrates a comparison of positions 122-181, 182-236, and237-277 of the Pp_PSE1 peptide sequence (SEQ ID NO:2) with positions41-96, 97-145, and 146-186 of the sequence of SEQ ID NO:4, respectively;

FIG. 6: illustrates a comparison of positions 189-205 of the Pp_PSE1peptide sequence (SEQ ID NO:2) with positions 174-190 of the sequence ofSEQ ID NO:6;

FIG. 7: illustrates a comparison of positions 127-184, and 185-203 ofthe Pp_PSE1 peptide sequence (SEQ ID NO:2) with positions 33-87, and88-106 of the sequence of the sequence of Cc_PSE1 (SEQ ID NO:8),respectively;

FIG. 8: illustrates a comparison of positions 1-49, 50-95, 96-143,144-192, 193-240, 241-263, and 264-290 of the Pp_PSE1 peptide sequence(SEQ ID NO:2) with positions 1-36, 37-82, 83-131, 132-177, 178-220,221-270, and 271-297 of the sequence of Tc_PSE2 (SEQ ID NO:6),respectively;

FIG. 9: illustrates a comparison of positions 6-55, 56-105, 106-145, and146-180 of the Cc_PSE1 peptide sequence (SEQ ID NO:8) with positions90-137, 138-186, 187-234, and 235-283 of the sequence of Tc_PSE2 (SEQ IDNO:6), respectively; and

FIG. 10: illustrates sequence motifs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides PSE nucleic acids and PSE proteinmolecules which participate in the metabolism of lipids and fatty acids,PUFA cofactors and enzymes in the moss Physcomitrella patens,Phytophthora infestans, Crypthecodinium or Traustochytrium or in thetransport of lipophilic compounds via membranes. The compounds accordingto the invention can be used for modulating the production of finechemicals from organisms, for example microorganisms, such as ciliates,fungi, yeasts, bacteria, algae, and/or plants such as maize, wheat, rye,oats, triticale, rice, barley, soybean, peanut, cotton, Brassicaspecies, such as oilseed rape, canola and turnip rape, pepper,sunflower, borage, evening primrose and tagetes, Solanaceae plants suchas potato, tobacco, aubergine and tomato, Vicia species, pea, cassava,alfalfa, shrub plants (coffee, cacao, tea), Salix species, trees (oilpalm, coconut) and perennial grasses and fodder crops, either directly(for example when the overexpression or optimization of a fatty acidbiosynthesis protein has a direct effect on the yield, production and/orproduction efficiency of the fatty acid from modified organisms) or theycan have an indirect effect which nevertheless leads to an increasedyield, production and/or production efficiency of a desired compound orto a decrease in undesired compounds (for example when the modulation ofthe lipid and fatty acid, cofactor and enzyme metabolism leads tochanges in yield, production and/or production efficacy or in thecomposition of the desired compounds within the cells, which, in turn,may affect the production of one or more fine chemicals). Aspects of theinvention are illustrated in greater detail hereinbelow.

I. Fine Chemicals and PUFAs

The term “fine chemicals” is known in the art and encompasses moleculeswhich have been produced by an organism and which are used in a varietyof industries such as, by way of example but not by way of limitation,the pharmaceuticals industry, agroindustry, food industry and cosmeticsindustry. These compounds encompass lipids, fatty acids, cofactors andenzymes and the like (as described, for example, in Kuninaka, A. (1996)Nucleotides and related compounds, pp. 561-612, in Biotechnology Vol. 6,Rehm et al., Ed.: VCH Weinheim and references cited therein), lipids,saturated and unsaturated fatty acids (for example arachidonic acid),vitamins and cofactors (as described in Ullmann's Encyclopedia ofIndustrial Chemistry, Vol. A27, Vitamins, pp. 443-613 (1996): VCHWeinheim, and references cited therein; and Ong, A. S., Niki, E., &Packer, L. (1995) Nutrition, Lipids, Health and Disease Proceedings ofthe UNESCO/Confederation of Scientific and Technological Associations inMalaysia and the Society for Free Radical Research—Asia, held Sep. 1-3,1994 in Penang, Malaysia, AOCS Press (1995)), enzymes and all otherchemicals described by Gutcho (1983) in Chemicals by Fermentation, NoyesData Corporation, ISBN: 0818805086 and references cited therein. Themetabolism and the uses of certain fine chemicals are illustrated ingreater detail hereinbelow.

The combination of various precursor molecules and biosynthetic enzymesleads to the production of various fatty acid molecules, which has adecisive effect on membrane composition. It can be assumed that PUFAsare not only just incorporated into triacylglycerol, but also intomembrane lipids.

Membrane synthesis is a well characterized process in which a number ofcomponents, inclusive of lipids as part of the bilayer membrane, areinvolved. The production of novel fatty acids such as PUFAs cantherefore generate novel properties of membrane functions within a cellor an organism.

Cell membranes serve a multiplicity of functions in a cell. First andforemost, a membrane delimits the contents of a cell from theenvironment, thus imparting integrity to the cell. Membranes can alsoact as barriers against the influx of dangerous or undesired compoundsor else against the efflux of desired compounds.

For more detailed descriptions of involvements of membranes and themechanisms involved, see Bamberg, E., et al. (1993) Charge transport ofion pumps on lipid bilayer membranes, Q. Rev. Biophys. 26:1-25; Gennis,R. B. (1989) Pores, Channels and Transporters, in: Biomembranes,Molecular Structure and Function, Springer: Heidelberg, pp. 270-322; andNikaido, H., and Saier, H. (1992) Transport proteins in bacteria: commonthemes in their design, Science 258:936-942, and the citations containedin each of these references.

Lipid synthesis can be divided into two parts: the synthesis of fattyacids and their binding to sn-glycerol-3-phosphate, and the addition ormodification of a polar head group. Customary lipids used in membranesencompass phospholipids, glycolipids, sphingolipids andphosphoglycerides. Fatty acid synthesis starts with the conversion ofacetyl-CoA either into malonyl-CoA by acetyl-CoA carboxylase or intoacetyl-ACP by acetyl transacylase. After a condensation reaction, thesetwo product molecules together form acetoacetyl-ACP, which is convertedvia a series of condensation, reduction and dehydration reactions togive a saturated fatty acid molecule with the desired chain length. Theproduction of the unsaturated fatty acids from these molecules iscatalyzed by specific desaturases, either aerobically by means ofmolecular oxygen or anaerobically (as regards fatty acid synthesis inmicroorganisms, see F. C. Neidhardt et al. (1996) E. coli andSalmonella. ASM Press: Washington, D.C., pp. 612-636 and referencescontained therein; Lengeler et al. (Ed.) (1999) Biology of Procaryotes.Thieme: Stuttgart, New York, and the references contained therein, andMagnuson, K., et al. (1993) Microbiological Reviews 57:522-542 and thereferences contained therein).

Examples of precursors for PUFA biosynthesis are linolic and linolenicacid. These C₁₈ carbon fatty acids must be elongated to C₂₀ or C₂₂ togive fatty acids of the eicosa and docosa chain type. Variousdesaturases such as enzymes which have Δ6 desaturase, Δ5- andΔ4-desaturase activity can lead to arachidonic acid, eicosapentaenoicacid and docosahexaenoic acid and various other long-chain PUFAs whichcan be extracted and used for various purposes in food and feed,cosmetic or pharmaceutical applications.

To produce long chain PUFAs, the polyunsaturated C₁₆- or C₁₈- orC₂₀-fatty acids must, as mentioned above, be elongated by at least twocarbon atoms by the enzymatic activity of an elongase. The nucleic acidsequences according to the invention encode first microbial elongasesfrom typical producers containing PUFA in the triacylglycerol fraction,which elongases are capable of elongating the C₁₆- or C₁₈- or C₂₀-fattyacids with at least two double bonds in the fatty acid by at least twocarbon atoms or which convert these, for example sequentially insuccession, by converting a C₁₆- or C₁₈-fatty acid into a C₂₀-fatty acidand then a C₂₀- into a C₂₂- or higher even numbered fatty acidcontaining units with 2 C atoms. After one elongation cycle, this enzymeactivity leads to C₂₀-fatty acids, and after two, three and fourelgonation cycles to C₂₂-, C₂₄- or C₂₆-fatty acids. Longer PUFAs canalso be synthesized with the elongase according to the invention. Theactivity of the elongases according to the invention preferably leads toC₂₀- and/or C₂₂-fatty acids with at least two double bonds in the fattyacid molecule, C₂₀-fatty acids, preferably with three, four or fivedouble bonds, especially preferably three double bonds, in the fattyacid molecule, C₂₂-fatty acids, preferably with three, four, five or sixdouble bonds, especially preferably five or six double bonds, in thefatty acid molecule. After elongation with the enzyme according to theinvention, further desaturation steps may be carried out. Thus, theproducts of the elongase activities and of the further desaturationwhich is possible lead to preferred PUFAs with a higher degree ofdesaturation, such as docosadienoic acid, arachidonic acid,ω6-eicosatrienedihomo-γ-linolenic acid, eicosapentaenoic acid,ω3-eicosatrienoic acid, ω3-eicosatetraenoic acid, docosapentaenoic acidor docosahexaenoic acid. Examples of substrates of this enzyme activityaccording to the invention are taxol acid, 7,10,13-hexadecatrienoicacid, 6,9-octadecadienoic acid, linolic acid, γ-linolenic acid,pinolenic acid, α-linolenic acid, arachidonic acid, eicosapentaenoicacid or stearidonic acid. Preferred substrates are linolic acid,γ-linolenic acid and/or α-linolenic acid or arachidonic acid,eicosatetraenoic acid or eicosapentaenoic acid. The C₁₆- or C₁₈- orC₂₀-fatty acids with at least two double bonds in the fatty acid can beelongated by the enzyme activity according to the invention in the formof the free fatty acid or in the form of the esters, such asphospholipids, glycolipids, sphingolipids, phosphoglycerides,monoacylglycerol, diacylglycerol or triacylglycerol.

Furthermore, fatty acids must subsequently be transported to variouslocations and incorporated into the triacylglycerol storage lipid.Another important step in lipid synthesis is the transfer of fatty acidsto the polar head groups, for example by glycerol fatty acidacyltransferase (see Frentzen, 1998, Lipid, 100(4 5):161-166).

For publications on plant fatty acid biosynthesis, desaturation, lipidmetabolism and the membrane transport of fatty compounds,beta-oxidation, fatty acid modification and cofactors, triacylglycerolstorage and assembly inclusive of the references cited therein, see thefollowing articles: Kinney, 1997, Genetic Engineering, Ed.: J K Setlow,19:149-166; Ohlrogge and Browse, 1995, Plant Cell 7:957-970; Shanklinand Cahoon, 1998, Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:611-641;Voelker, 1996, Genetic Engineering, Ed.: JK Setlow, 18:111-13; Gerhardt,1992, Prog. Lipid R. 31:397-417; Gühnemann Schäfer & Kindl, 1995,Biochim. Biophys Acta 1256:181-186; Kunau et al., 1995, Prog. Lipid Res.34:267-342; Stymne et al., 1993, in: Biochemistry and Molecular Biologyof Membrane and Storage Lipids of Plants, Ed.: Murata and Somerville,Rockville, American Society of Plant Physiologists, 150 158, Murphy &Ross 1998, Plant Journal. 13(1):1-16.

Vitamins, cofactors and “nutraceuticals”, such as PUFAs, encompass agroup of molecules which higher animals can no longer synthesize andtherefore have to take up, or which higher animals can no longersynthesize themselves to a sufficient degree and must therefore take upadditionally, even though they are readily synthesized by otherorganisms such as bacteria. The biosynthesis of these molecules inorganisms which are capable of producing them, such as in bacteria, hasbeen more or less characterized (Ullmann's Encyclopedia of IndustrialChemistry, “Vitamins”, Vol. A27, pp. 443-613, VCH Weinheim, 1996;Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry andMolecular Biology, John Wiley & Sons; Ong, A. S., Niki, E., & Packer, L.(1995) “Nutrition, Lipids, Health and Disease” Proceedings of theUNESCO/Confederation of Scientific and Technological Associations inMalaysia and the Society for Free Radical Research Asia, held Sep. 1-3,1994, in Penang, Malaysia, AOCS Press, Champaign, Ill. X, 374 pp).

The abovementioned molecules are either biologically active moleculesthemselves or precursors of biologically active substances which acteither as electron carriers or as intermediates in a multiplicity ofmetabolic pathways. Besides their nutritional value, these compoundsalso have a significant industrial value as colorants, antioxidants andcatalysts or other processing auxiliaries. (For a review over structure,activity and industrial applications of these compounds, see, forexample, Ullmann's Encyclopedia of Industrial Chemistry, “Vitamins”,Vol. A27, pp. 443-613, VCH Weinheim, 1996). Polyunsaturated fatty acidshave a variety of functions and health promoting effects, for example inthe case of coronary heart disease, inflammatory mechanisms, children'snutrition and the like. For publications and references including thereferences cited therein, see: Simopoulos, 1999, Am. J. Clin. Nutr. 70(3rd Suppl.):560-569, Takahata et al., Biosc. Biotechnol. Biochem. 1998,62(11):2079-2085, Willich and Winther, 1995, Deutsche MedizinischeWochenschrift 120(7):229 et seq.

II. Elements and Processes of the Invention

The present invention is based at least in part on the discovery ofnovel molecules termed herein PSE nucleic acid and PSE proteinmolecules, which exert an effect on the production of cell membranes inPhyscomitrella patens, Crypthecodinium cohnii, Phytophthora infestans,Thraustochytrium and/or Ceratodon purpureus and, for example, have aneffect on the movement of molecules via these membranes. In oneembodiment, the PSE molecules participate in the metabolism of compoundsrequired for the synthesis of cell membranes in organisms such asmicroorganisms and plants or indirectly affect the transport ofmolecules via these membranes. In a preferred embodiment, the activityof the PSE molecules according to the invention for regulating theproduction of membrane components and membrane transport has an effecton the production of the desired fine chemical by this organism. In anespecially preferred embodiment, the activity of the PSE moleculesaccording to the invention is modulated so that the yield, productionand/or production efficiency of the metabolic pathways of microorganismsor plants which regulate the PSEs according to the invention aremodulated and the transport efficiency of compounds through themembranes is modified, which either directly or indirectly modulates theyield, production and/or production efficiency of a desired finechemical by microorganisms and plants.

The term PSE or PSE polypeptide encompasses proteins which participatein the metabolism of compounds required for the synthesis of cellmembranes in organisms such as microorganisms and plants or in thetransport of molecules via these membranes. Examples of PSEs aredisclosed in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9 and SEQ ID NO:11 or their homologs, derivatives or analogs. Theterms PSE or PSE nucleic acid sequence(s) encompass nucleic acidsequences which encode a PSE and part of which can be a coding regionand also corresponding 5′- and 3′-untranslated sequence regions.Examples of PSE genes are the sequences shown in SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11. The termsproduction and productivity are known in the art and encompass theconcentration of the fermentation product (for example of the desiredfine chemical) which is formed within a certain period and in a certainfermentation volume (for example kg product per hour per liter). Theterm production efficiency encompasses the time required for achieving aparticular product quantity (for example the time required by the cellto establish a particular throughput rate of a fine chemical). The termyield or product/carbon yield is known in the art and encompasses theefficiency with which the carbon source is converted into the product(i.e. the fine chemical). This is usually expressed as, for example, kgproduct per kg carbon source. Increasing the yield or production of thecompound increases the amount of the molecules obtained or of thesuitable molecules of this compound obtained in a specific quantity ofculture over a defined period. The terms biosynthesis or biosyntheticpathway are known in the art and encompass the synthesis of a compound,preferably of an organic compound, by a cell from intermediates, forexample in a multi-step process which is subject to strong regulation.The terms catabolism or catabolic pathway are known in the art andencompass the cleavage of a compound, preferably of an organic compound,by a cell into catabolites (in general smaller or less complexmolecules), for example in a multi-step process which is subject tostrong regulation. The term metabolism is known in the art andencompasses the totality of the biochemical reactions which take placein an organism. The metabolism of a certain compound (for example themetabolism of a fatty acid) thus encompasses the totality of thebiosynthetic, modification and catabolic pathways of this compound inthe cell which are relevant to this compound.

In another embodiment, the PSE molecules according to the invention canmodulate the production of a desired molecule, such as a fine chemical,in a microorganism or in plants. There exists a series of mechanisms bywhich the modification of a PSE according to the invention can directlyaffect the yield, production and/or production efficiency of a finechemical from a microorganism strain or plant strain comprising thismodified protein. The number or activity of PSEs participating in thetransport of molecules of fine chemicals within, or out of, the cell canbe increased, so that greater amounts of these compounds are transportedvia membranes, from which they can be obtained and converted into eachother with greater ease. Furthermore, fatty acids, triacylglycerolsand/or lipids are desirable fine chemicals themselves; optimizing theactivity or increasing the number of one or more PSEs according to theinvention which participate in the biosynthesis of these compounds, orby interfering with the activity of one or more PSEs which participatein the catabolism of these compounds makes increasing the yield,production and/or production efficiency of fatty acid molecules andlipid molecules from organisms such as microorganisms or plants,possible.

The mutagenesis of the PSE gene according to the invention can also giverise to PSEs with modified activities which indirectly affect theproduction of one or more desired fine chemicals from microorganisms orplants. For example, PSEs according to the invention which participatein the export of waste products can exhibit a greater number or higheractivity, so that the normal metabolic waste products of the cell (whosequantity might be increased owing to the overproduction of the desiredfine chemical) are exported efficiently before they can damage themolecules in the cell (which would reduce the cell's viability) orinterfere with the biosynthetic pathways of the fine chemicals (whichwould reduce the yield, production or production efficiency of a desiredfine chemical). The relatively large intracellular amounts of thedesired fine chemical themselves can furthermore be toxic to the cell,so that increasing the activity or number of transporters capable ofexporting these compounds from the cell results in an increasedviability of the cell in culture, which, in turn, leads to a highernumber of cells in the culture which produce the desired fine chemical.The PSEs according to the invention can also be manipulated in such away that the corresponding amounts of different lipid molecules andfatty acid molecules are produced. This can have a substantial effect onthe lipid composition of the cell membrane. Since each lipid type hasdifferent physical properties, a modification of the lipid compositionof a membrane can significantly modify membrane fluidity. Modificationsof the membrane fluidity can affect the transport of molecules via themembrane and cell integrity, each of which has a substantial effect onthe production of fine chemicals from microorganisms and plants in largescale fermentation culture. Plant membranes impart specific propertiessuch as tolerance to high and low temperatures, salt, drought andtolerance with respect to pathogens such as bacteria and fungi. Themodulation of the membrane components may therefore have a criticaleffect on the ability of the plants to survive under the abovementionedstress parameters. This can take place via changes in signal cascades ordirectly via the modified membrane composition (see, for example,Chapman, 1998, Trends in Plant Science, 3(11):419-426) and signalcascades (see Wang 1999, Plant Physiology, 120:645-651) or affect thetolerance of low temperatures, as disclosed in WO 95/18222.

The isolated nucleic acid sequences according to the invention arepresent, for example, in the genome of a Thraustochytrium strain whichis available via the American Type Culture Collection (ATCC) with thestrain number ATCC26185 (Thraustochytrium), or, in the case ofCrypthecodinium, for example, accessible via the Provasoli-GuillardNational Center for Culture of Marine Phytoplankton ((CCMP) WestBoothbay Harbour, Me., USA) with the strain culture No. CCMP316. In thecase of Phytophthora infestans, the stated nucleic acid molecules areisolated from the strain ATCC 48886.

The nucleotide sequence of the isolated Physcomitrella, Crypthecodinium,Phytophthora infestans or Thraustochytrium cDNA and the deduced aminoacid sequences of the Physcomitrella patens PSEs are shown in SEQ IDNO:1 to SEQ ID NO:12. Computer analyses were carried out which classifyand/or identify these nucleotide sequences as sequences which encodeproteins participating in the metabolism of cell membrane components orwhich participate in the transport of compounds via cell membranes, orof PUFA biosynthesis. ESTs with the database input No. PP001019019F,CC001042041R, PI001002014R, TC002034029R, TC002034029R-11 andTC002014093R in the database of the inventor constitute the sequencesaccording to the invention in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQID NO:7, SEQ ID NO:9 and SEQ ID NO:11. In a similar manner, the partialpolypeptides were termed PP001019019F, CC001042041R, PI001002014R,TC002034029R, TC002034029R-11 and TC002014093R and constitute thesequences according to the invention in SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12 in accordance withTable 2. The complete fragment insert of the ESTs TC002034029R wassequenced and resulted in SEQ ID NO:3, which is the complete sequence ofTC002034029R. TC002034029R-11 describes a full length sequence of anelongase from Thraustochytrium. The naming of the remaining clones issimilar. Also, corresponding gene names were assigned to the variousclones. Abbreviations: Tc=Thraustochytrium, Cc=Crypthecodinium,Pp=Physcomitrella patens, P: Phytophthora infestans.

TABLE 2 Polypeptide Nucleic acid Name/EST name Gene name SEQ ID NO SEQID NO PP001019019F Pp_PSE1 2 1 TC002034029R Tc_PSE1 4 3 TC002014093RTc_PSE2 6 5 CC001042041R Cc_PSE1 8 7 TC002034029R-11 Tc_PSE1_1 10 9PI001002014R Pi_PSE1 12 11

The present invention also relates to proteins with an amino acidsequence which is essentially homologous with an amino acid sequence ofSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQID NO:12. As used in the present context, a protein with an amino acidsequence which is essentially homologous with a selected amino acidsequence has at least approximately 50% homology with the selected aminoacid sequence, for example the complete amino acid sequence selected. Aprotein with an amino acid sequence which is essentially homologous witha selected amino acid sequence can also have at least approximately 50to 60%, preferably at least approximately 60 to 70%, more preferably atleast approximately 70 to 80%, 80 to 90% or 90 to 95%, and mostpreferably at least approximately 96%, 97%, 98%, 99% or more homologywith a selected amino acid sequence.

The PSE according to the invention or the biologically active part orthe fragment thereof can participate in the metabolism of compoundsrequired for the synthesis of cell membranes in microorganisms or plantsor in the transport of molecules via these membranes or have one or moreof the activities required for the elongation of C₁₆- or C₁₈- orC₂₀-PUFAs, so that C₂₀-, C₂₂- or C₂₄-PUFAs and related PUFAs areobtained.

Various aspects of the invention are described in greater detail in thesubsections which follow.

a. Isolated Nucleic Acid Molecules

One embodiment of the invention comprises isolated nucleic acids derivedfrom PUFA producing microorganisms and encoding polypeptides whichelongate C₁₆- or C₁₈-fatty acids with at least two double bonds in thefatty acid by at least two carbon atoms or which elongate C₂₀-fattyacids with at least two double bonds in the fatty acid by at least twocarbon atoms.

A further embodiment according to the invention comprises isolatednucleic acids encompassing nucleotide sequences encoding polypeptideswhich elongate C₁₆-, C₁₈- or C₂₀-fatty acids with at least two doublebonds in the fatty acid and which are selected from the group consistingof

-   a) the nucleic acid sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ    ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11,-   b) a nucleic acid sequence which, in accordance with the degeneracy    of the genetic code, is derived from one of the sequences shown in    SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and    SEQ ID NO:11 or-   c) derivatives of the sequence shown in SEQ ID NO:1, SEQ ID NO:3,    SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11 which encode    polypeptides of the amino acid sequence shown in SEQ ID NO:2, SEQ ID    NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12 and    which have at least 50% homology at the amino acid level without the    enzymatic action of the polypeptides being substantially reduced.

The abovementioned nucleic acids according to the invention, which actas C₁₆-, C₁₈- or C₂₀-elongase, are derived from organisms such asciliates, fungi, algae, plants or dinoflagellates which are capable ofsynthesizing PUFAs, preferably from plants or algae, especiallypreferably from the genus Phytophthora, Physcomitrella, Crypthecodinium,Thraustochytrium or Schizochytrium, most preferably from Phytophthorainfestans, Physcomitrella patens, Crypthecodinium cohnii orThraustochytrium sp., Schizochytrium sp. or closely related organisms.

One aspect of the invention relates to isolated nucleic acid moleculeswhich encode PSE polypeptides or biologically active parts thereof, andto nucleic acid fragments which suffice for use as hybridization probesor primers for identifying or amplifying a PSE-encoding nucleic acid(for example PSE DNA). The term “nucleic acid molecule” as used in thepresent context is intended to encompass DNA molecules (for example cDNAor genomic DNA) and RNA molecules (for example mRNA) and DNA or RNAanalogs which are generated by means of nucleotide analogs. This termadditionally encompasses the untranslated sequence at the 3′ and the 5′end of the coding gene region: at least approximately 100 nucleotides ofthe sequence upstream of the 5′ end of the coding region and at leastapproximately 20 nucleotides of the sequence downstream of the 3′ end ofthe coding gene region. The nucleic acid molecule can be single- ordouble-stranded, but is preferably double-stranded DNA. An “isolated”nucleic acid molecule is separated from other nucleic acid moleculeswhich are present in the natural source of the nucleic acid. An“isolated” nucleic acid preferably has no sequences which naturallyflank the nucleic acid in the genomic DNA of the organism from which thenucleic acid is derived (for example sequences located at the 5′ and 3′ends of the nucleic acid). In various embodiments, the isolated PSEnucleic acid molecule can contain, for example, less than approximately5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb nucleotide sequenceswhich naturally flank the nucleic acid molecule in the genomic DNA ofthe cell from which the nucleic acid is derived (for example aPhyscomitrella patens cell). An “isolated” nucleic acid molecule, suchas a cDNA molecule, can moreover be essentially free from other cellularmaterial or culture medium if it is generated by recombinant techniques,or free from chemical precursors or other chemicals if it is synthesizedchemically.

A nucleic acid molecule according to the invention, for example anucleic acid molecule with a nucleotide sequence of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11 or a partthereof, can be isolated using standard techniques of molecular biologyand the sequence information provided herein. Also, for example ahomologous sequence or homologous, conserved sequence regions at the DNAor amino acid level can be identified with the aid of alignmentalgorithms. For example, a Phytophthora, Physcomitrella, Crypthecodiniumor Thraustochytrium cDNA can be isolated from a Phytophthora,Physcomitrella, Crypthecodinium or Thraustochytrium library by using thecomplete SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9and/or SEQ ID NO:11 or a part thereof as hybridization probe andstandard hybridization techniques (such as, for example, as described inSambrook et al., Molecular Cloning: A Laboratory Manual. 2nd Ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989). Moreover, a nucleic acid moleculeencompassing a complete sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11 or a part thereof can beisolated by polymerase chain reaction, where oligonucleotide primerswhich are generated on the basis of this sequence or parts thereof, inparticular regions around His-box motifs of SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12 or modificationsof the same in individual, defined amino acids are used (for example, anucleic acid molecule encompassing the complete sequence of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11 or apart thereof can be isolated by polymerase chain reaction usingoligonucleotide primers which have been generated on the basis of thissame sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQID NO:9 and SEQ ID NO:11). Furthermore, especially suitable for thispurpose are those partial sequences as they are shown in FIG. 10. Forexample, mRNA can be isolated from cells (for example by the guanidiniumthiocyanate extraction method by Chirgwin et al. (1979) Biochemistry18:5294-5299), and cDNA can be generated by means of reversetranscriptase (for example Moloney MLV reverse transcriptase, availablefrom Gibco/BRL, Bethesda, Md., or AMV reverse transcriptase, availabefrom Seikagaku America, Inc., St. Petersburg, Fla.). Syntheticoligonucleotide primers for amplification by means of polymerase chainreaction can be generated on the basis of one of the nucleotidesequences shown in SEQ ID NO:1, 3, 5, 7, 9 or 11 or with the aid of theamino acid sequences shown in FIG. 10. A nucleic acid according to theinvention can be amplified using cDNA or, alternatively, using genomicDNA as template and suitable oligonucleotide primers, in accordance withstandard PCR amplification techniques. The nucleic acid thus amplifiedcan be cloned into a suitable vector and characterized by means of DNAsequence analysis. Oligonucleotides which correspond to a PSE nucleotidesequence can be generated by standard synthesis methods, for examplewith an automatic DNA synthesizer.

The cDNA shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,SEQ ID NO:9 and SEQ ID NO:11 encompasses sequences which encode PSEs(i.e. the “coding region”) and also 5′-untranslated sequences and3′-untranslated sequences. Alternatively, the nucleic acid molecule canonly encompass the coding region of one of the sequences in SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11 orcan comprise complete genomic fragments isolated from genomic DNA.

SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQID NO:12 are identified by the same EST input number code as SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO and SEQ ID NO:11for ease of correlation.

In a further preferred embodiment, an isolated nucleic acid moleculeaccording to the invention encompasses a nucleic acid molecule which isa complement of one of the nucleotide sequences shown in SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO and SEQ ID NO:11 or apart thereof. A nucleic acid molecule which is complementary to one ofthe nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11 is sufficiently complementaryif it is capable of hybridizing with one of the sequences stated in SEQID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ IDNO:11, giving rise to a stable duplex.

Homologs of the new elongase nucleic acid sequences with the sequenceSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQID NO:11 means, for example, allelic variants with at leastapproximately 50 to 60%, preferably at least approximately 60 to 70%,more preferably at least approximately 70 to 80%, 80 to 90% or 90 to95%, and even more preferably at least approximately 95%, 96%, 97%, 98%,99% or more homology with one of the nucleotide sequences shown in SEQID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ IDNO:11 or their homologs, derivatives or analogs or parts thereof. In afurther preferred embodiment, an isolated nucleic acid moleculeaccording to the invention encompasses a nucleotide sequence whichhybridizes with one of the nucleotide sequences shown in SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO and SEQ ID NO:11 or apart thereof, under stringent conditions. Allelic variants encompass, inparticular, functional variants which can be obtained by the deletion,insertion or substitution of nucleotides from/into the sequence shown inSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQID NO:11, it being intended, however, for the enzyme activity of theresulting proteins which are synthesized to be advantageously retainedfor the insertion of one or more genes. Proteins which retain theenzymatic activity of elongase means proteins with at least 10%,preferably 20%, especially preferably 30%, very especially preferably40% of the original enzyme activity compared with the protein encoded bySEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQID NO:12. Elongases which retain the abovementioned activities areelongases whose enzymatic activity is not substantially reduced.

Homologs of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9 and SEQ ID NO:11 also means, for example, bacterial, fungal andplant homologs, truncated sequences, single-stranded DNA or RNA of thecoding and noncoding DNA sequence.

Homologs of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9 and SEQ ID NO:11 also means derivatives such as, for example,promoter variants. The promoters upstream of the nucleotide sequencesstated can be modified by one or more nucleotide substitutions, byinsertion(s) and/or deletion(s), without, however, interfering with thefunctionality or activity of the promoters. It is furthermore possiblefor the activity of the promoters to be increased by modification oftheir sequence or for them to be replaced completely by more activepromoters, even from heterologous organisms.

Moreover, the nucleic acid molecule according to the invention can onlyencompass part of the coding region of one of the sequences in SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ IDNO:11, for example a fragment which can be used as probe or primer or afragment which encodes a biologically active segment of a PSE. Thenucleotide sequences determined from cloning the PSE gene ofPhyscomitrella patens, Phytophthora infestans, Thraustochytrium andCrypthecodinium allow the generation of probes and primers which aredesigned for identifying and/or cloning PSE homologs in other cell typesand organisms and PSE homologs from other mosses or related species. Theprobe/primer normally encompasses essentially purified oligonucleotide.The oligonucleotide normally encompasses a nucleotide sequence regionwhich hybridizes under stringent conditions with at least approximately12, preferably approximately 16, more preferably approximately 25, 40,50 or 75 successive nucleotides of a sense strand of one of thesequences stated in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,SEQ ID NO and SEQ ID NO:11, of an antisense strand of one of thesequences stated in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,SEQ ID NO and SEQ ID NO or its homologs, derivatives and analogs ornaturally occurring mutants thereof. Primers based on a nucleotidesequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO and SEQ ID NO:11 can be used in PCR reactions for cloning PSEhomologs. Probes based on the PSE nucleotide sequences can be used fordetecting transcripts or genomic sequences which encode the same orhomologous proteins. In preferred embodiments, the probe additionallyencompasses a labeling group bound thereto, for example a radioisotope,a fluorescent compound, an enzyme or an enzyme cofactor. These probescan be used as part of a test kit for genomic markers for identifyingcells which misexpress a PSE, for example by measuring an amount of aPSE-encoding nucleic acid in a cell sample, for example measuring thePSE mRNA level, or for determining whether a genomic PSE gene is mutatedor deleted.

In one embodiment, the nucleic acid molecule according to the inventionencodes a protein or a part thereof which encompasses an amino acidsequence which has sufficient homology with an amino acid sequence ofSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQID NO:12 for the protein or the part thereof to retain the ability toparticipate in the metabolism of compounds required for the synthesis ofcell membranes in microorganisms or plants or in the transport ofmolecules via these membranes. As used in the present context, the term“sufficient homology” relates to proteins or parts thereof whose aminoacid sequences have a minimum number of amino acid residues (for examplean amino acid residue with a similar side chain, such as an amino acidresidue in one of the sequences of SEQ ID NO:2 to 12) which areidentical with or equivalent to an amino acid sequence of SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12 sothat the protein or the part thereof can participate in the metabolismof compounds required for the synthesis of cell membranes inmicroorganisms or plants or in the transport of molecules via thesemembranes. As described herein, protein components of these metabolicpathways for membrane components or membrane transport systems can playa role in the production and secretion of one or more fine chemicals.Examples of these activities are also described herein. Thus, the“function of a PSE” contributes either directly or indirectly to theyield, production and/or production efficiency of one or more finechemicals. Examples of PSE substrate specificities of the catalyticactivity are stated in Table 1.

In a further embodiment, derivatives of the nucleic acid moleculeaccording to the invention encode proteins with at least approximately50 to 60%, preferably at least approximately 60 to 70% and morepreferably at least approximately 70 to 80%, 80 to 90%, 90 to 95% andmost preferably at least approximately 96%, 97%, 98%, 99% or morehomology with a complete amino acid sequence of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12. Thehomology of the amino acid sequence was determined over the entiresequence region using the program PileUp (J. Mol. Evolution., 25,351-360, 1987, Higgins et al., CABIOS, 5, 1989:151-153) or BESTFIT orGAP (Henikoff, S, and Henikoff, J. G. (1992). Amino acid substitutionmatrices from protein blocks. Proc. Natl. Acad. Sci. USA 89:10915-10919.)

Parts of proteins encoded by the PSE nucleic acid molecules according tothe invention are preferably biologically active parts of one of thePSEs. As used herein, the term “biologically active part of a PSE” isintended to encompass a segment, for example a domain/motif, of a PSEwhich can participate in the metabolism of compounds required for thesynthesis of cell membranes in microorganisms or plants or in thetransport of molecules via these membranes or which has an activitystated in Table 1. An assay of the enzymatic activity can be carried outin order to determine whether a PSE or a biologically active partthereof can participate in the metabolism of compounds required for thesynthesis of cell membranes in microorganisms or plants or in thetransport of molecules via these membranes. These assay methods asdescribed in detail in Example 8 of the examples section are known tothe skilled worker.

Additional nucleic acid fragments which encode biologically activesegments of a PSE can be generated by isolating part of one of thesesequences in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10 and SEQ ID NO:12, expressing the encoded segment of the PSE or ofthe peptide (for example by recombinant expression in vitro) anddetermining the activity of the encoded part of the PSE or of thepeptide.

Moreover, the invention encompasses nucleic acid molecules which differfrom one of the nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11 (and partsthereof) owing to the degeneracy of the genetic code and which thusencode the same PSE as the one encoded by the nucleotide sequences shownin SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 andSEQ ID NO:11. In another embodiment, an isolated nucleic acid moleculeaccording to the invention has a nucleotide sequence which encodes aprotein with an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12. In a furtherembodiment, the nucleic acid molecule according to the invention encodesa full-length PSE protein which is essentially homologous with an aminoacid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10 and SEQ ID NO:12 (which is encoded by an open reading frameshown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9and SEQ ID NO:11) and which can be identified and isolated by customarymethods.

In addition to the PSE nucleotide sequences shown in SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11, theskilled worker recognizes that DNA sequence polymorphisms may existwhich lead to changes in the amino acid sequences of the PSEs within apopulation (for example the Physcomitrella, Phytophthora,Crypthecodinium or Thraustochytrium population). These geneticpolymorphisms in the PSE gene can exist between individuals within apopulation owing to natural variation. As used in the present context,the terms “gene” and “recombinant gene” refer to nucleic acid moleculeswith an open reading frame which encodes a PSE, preferably aPhytophthora, Physcomitrella, Crypthecodinium or Thraustochytrium PSE.These natural variants usually cause a variance of 1 to 5% in thenucleotide sequence of the PSE gene. All of these nucleotide variationsand resulting amino acid polymorphisms in PSE which are the result ofnatural variation and do not alter the functional activity of PSEs areintended to come within the scope of the invention.

Nucleic acid molecules which correspond to the natural variants andnon-Physcomitrella, -Phytophthora, -Crypthecodinium or -Thraustochytriumhomologs, derivatives and analogs of the Phytophthora, Physcomitrella,Crypthecodinium or Thraustochytrium cDNA can be isolated in accordancewith standard hybridization techniques under stringent hybridizationconditions owing to their homology with the Phytophthora,Physcomitrella, Crypthecodinium or Thraustochytrium PSE nucleic aciddisclosed herein using the Physcomitrella, Phytophthora, Crypthecodiniumor Thraustochytrium cDNA or a part thereof as hybridization probe. Inanother embodiment, an isolated nucleic acid molecule according to theinvention has a minimum length of 15 nucleotides and hybridizes understringent conditions with the nucleic acid molecule which encompasses anucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9 and SEQ ID NO:11. In other embodiments, the nucleicacid has a minimum length of 25, 50, 100, 250 or more nucleotides. Theterm “hybridizes under stringent conditions” as used in the presentcontext is intended to describe hybridization and wash conditions underwhich nucleotide sequences which have at least 60% homology with eachother usually remain hybridized with each other. The conditions arepreferably such that sequences which have at least approximately 65%,more preferably at least approximately 70% and even more preferably atleast approximately 75% or more homology with each other usually remainhybridized with each other. These stringent conditions are known to theskilled worker and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred,nonlimiting example of stringent hybridization conditions arehybridizations in 6× sodium chloride/sodium citrate (sodiumchloride/sodium citrate=SSC) at approximately 45° C. followed by one ormore wash steps in 0.2×SSC, 0.1% SDS at 50 to 65° C. It is known to theskilled worker that these hybridization conditions differ depending onthe type of the nucleic acid and, for example when organic solvents arepresent, with regard to buffer temperature and concentration. Forexample, the temperature differs under “standard hybridizationconditions” depending on the type of the nucleic acid between 42° C. and58° C. in aqueous buffer with a concentration of 0.1 to 5×SSC (pH 7.2).If organic solvent is present in the abovementioned buffer, for example50% formamide, the temperature under standard conditions isapproximately 42° C. The hybridization conditions for DNA:DNA hybridsare preferably for example 0.1×SSC and 20° C. to 45° C., preferablybetween 30° C. and 45° C. The hybridization conditions for DNA:RNAhybrids are preferably for example 0.1×SSC and 30° C. to 55° C.,preferably between 45° C. and 55° C. The abovementioned hybridizationtemperatures are determined for example for a nucleic acid approximately100 bp (=base pairs) in length and a G+C content of 50% in the absenceof formamide. The skilled worker knows how the hybridization conditionsrequired can be determined with reference to textbooks, such as the onementioned above or from the following textbooks: Sambrook et al.,“Molecular Cloning”, Cold Spring Harbor Laboratory, 1989; Hames andHiggins (Ed.) 1985, “Nucleic Acids Hybridization: A Practical Approach”,IRL Press at Oxford University Press, Oxford; Brown (Ed.) 1991,“Essential Molecular Biology: A Practical Approach”, IRL Press at OxfordUniversity Press, Oxford.

Preferably, an isolated nucleic acid molecule according to the inventionwhich hybridizes under stringent conditions with a sequence of SEQ ID NOSEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO and SEQ ID NO:11corresponds to a naturally occurring nucleic acid molecule. As used inthe present context, a “naturally occurring” nucleic acid moleculerefers to an RNA or DNA molecule with a nucleotide sequence which occursin nature (for example which encodes a natural protein). In oneembodiment, the nucleic acid encodes a naturally occurringPhyscomitrella patens PSE, Phytophthora infestans PSE, Crypthecodiniumcohnii PSE or Thraustochytrium PSE.

In addition to naturally occurring variants of the PSE sequence whichmay exist in the population, the skilled worker furthermore recognizesthat changes by means of mutation may also be introduced into anucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9 and SEQ ID NO:11, which leads to changes in the aminoacid sequence of the encoded PSE without adversely affecting thefunctionality of the PSE protein. For example, nucleotide substitutionswhich lead to amino acid substitutions on “nonessential” amino acidresidues can be generated in a sequence of SEQ ID NO:1, SEQ ID NO:3, SEQID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11. A “nonessential”amino acid residue is a residue which can be altered in a wild-typesequence of one of the PSEs (SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8, SEQ ID NO:10 and SEQ ID NO:12) without altering the activity ofthe PSE, while an “essential” amino acid residue is required for the PSEactivity. Other amino acid residues (for example those which are notconserved, or only semi conserved, in the domain with PSE activity),however, may not be essential for the activity and can thereforeprobably be altered without altering the PSE activity.

Accordingly, a further aspect of the invention relates to nucleic acidmolecules which encode PSEs comprising altered amino acid residues whichare not essential for the PSE activity. These PSEs differ from asequence in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10 and SEQ ID NO:12 with regard to the amino acid sequence whilestill retaining at least one of the PSE activities described herein. Inone embodiment, the isolated nucleic acid molecule encompasses anucleotide sequence encoding a protein, the protein encompassing anamino acid sequence with at least approximately 50% homology with anamino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10 and SEQ ID NO:12 and being able to participate in themetabolism of compounds required for the synthesis of cell membranes inPhytophthora, Physcomitrella, Crypthecodinium or Thraustochytrium or inthe transport of molecules via these membranes. The protein encoded bythe nucleic acid molecule preferably has at least approximately 50 to60% homology with one of the sequences in SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12, more preferably atleast approximately 60 to 70% homology with one of the sequences in SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ SEQ ID NO and SEQ ID NO:12, evenmore preferably at least approximately 70 to 80%, 80 to 90%, 90 to 95%homology with one of the sequences in SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12, and most preferably atleast approximately 96%, 97%, 98% or 99% homology with one of thesequences in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10 and SEQ ID NO:12.

To determine the percentage homology of two amino acid sequences (forexample one of the sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO and a mutated form thereof) orof two nucleic acids, the sequences are written one underneath the otherto allow optimal comparison (for example, gaps may be introduced intothe sequence of a protein or of a nucleic acid in order to generate anoptimal alignment with the other protein or the other nucleic acid).Then, the amino acid residues or nucleotides on the corresponding aminoacid positions or nucleotide positions are compared. If a position in asequence (for example one of the sequences of SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12) is occupied bythe same amino acid residue or the same nucleotide as the correspondingposition in the other sequence (for example a mutated form of thesequence selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10 and SEQ ID NO:12), then the molecules are homologousat this position (i.e. amino acid or nucleic acid “homology” as used inthe present context corresponds to amino acid or nucleic acid“identity”). The percentage homology between the two sequences is afunction of the number of identical positions which the sequences share(i.e. % homology=number of identical positions/total number of positions×100).

An isolated nucleic acid molecule which encodes a PSE which ishomologous with a protein sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12 can be generated byintroducing one or more nucleotide substitutions, additions or deletionsinto a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQID NO:7, SEQ ID NO:9 and SEQ ID NO:11 so that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedprotein. Mutations can be introduced into one of the sequences of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ IDNO:11 by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. Preferably, conservative amino acidsubstitutions are generated at one or more of the predicted nonessentialamino acid residues. In a “conservative amino acid substitution”, theamino acid residue is exchanged for an amino acid residue with a similarside chain. Families of amino acid residues with similar side chainshave been defined in the specialist field. These families encompassamino acids with basic side chains (for example lysine, arginine,histidine), acidic side chains (for example aspartic acid, glutamicacid), uncharged polar side chains (for example glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), unpolar side chains,(for example alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan), beta branched side chains (forexample threonine, valine, isoleucine) and aromatic side chains (forexample tyrosine, phenylalanine, tryptophan, histidine). A predictednonessential amino acid residue in a PSE is thus preferably exchangedfor another amino acid residue from the same side-chain family. As analternative, in another embodiment, the mutations can be introducedrandomly over all or part of the PSE-encoding sequence, for example bysaturation mutagenesis, and the resulting mutants can be screened forthe PSE activity described herein in order to identify mutants whichretain PSE activity. After the mutagenesis of one of the sequences ofSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQID NO:11, the encoded protein can be expressed recombinantly, and theactivity of the protein can be determined, for example using the assaydescribed herein (see examples section).

In addition to the nucleic acid molecules which encode the abovedescribed PSEs, a further aspect of the invention relates to isolatednucleic acid molecules which are “antisense” thereto. An “antisense”nucleic acid encompasses a nucleotide sequence which is complementary toa “sense” nucleic acid which encodes a protein, for examplecomplementary to the coding strand of a double stranded cDNA molecule orcomplementary to an mRNA sequence. Accordingly, an antisense nucleicacid can bind to a sense nucleic acid via hydrogen bonds. The antisensenucleic acid can be complementary to a complete PSE-encoding strand oronly to part thereof. In one embodiment, an antisense nucleic acidmolecule is “antisense” to a “coding region” of the coding strand of anucleotide sequence encoding a PSE. The term “coding region” refers tothe region of the nucleotide sequence which encompasses codons which aretranslated into amino acid residues (for example the entire codingregion which starts and ends with the stop codon, i.e. the last codonbefore the stop codon). In a further embodiment, the antisense nucleicacid molecule is “antisense” to a “noncoding region” of the codingstrand of a nucleotide sequence encoding PSE. The term “noncodingstrand” refers to 5′ and 3′ sequences which flank the coding region andare not translated into amino acids (i.e. which are also termed 5′- and3′-untranslated regions).

Taking into consideration the PSE-encoding sequences disclosed herein ofthe coding strand (for example the sequences shown in SEQ ID NO:1, SEQID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11),antisense nucleic acids according to the invention can be designed inaccordance with the rules of the Watson Crick base pairing. Theantisense nucleic acid molecule can be complementary to all of thecoding region of PSE mRNA, but is more preferably an oligonucleotidewhich is “antisense” to only part of the coding or noncoding region ofPSE mRNA. For example, the antisense oligonucleotide can becomplementary to the region around the translation start of PSE mRNA. Anantisense oligonucleotide can have a length of, for example,approximately 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 and morenucleotides. An antisense oligonucleotide is advantageously 15 to 25nucleotides in length. An antisense nucleic acid according to theinvention can be constructed by processes known in the art usingchemical synthesis and enzymatic ligation reactions. For example, anantisense nucleic acid (for example an antisense oligonucleotide) can besynthesized chemically, making use of naturally occurring nucleotides orvarious modified nucleotides which are such that they increase thebiological stability of the molecules or increase the physical stabilityof the duplex formed between the antisense and the sense nucleic acid;for example, phosphorothioate derivatives and acridin-substitutednucleotides may be used. Examples of modified nucleotides which may beused for generating the antisense nucleic acid are, inter alia,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthin, xanthin, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl 2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil,5-methoxyuracil, 2-methylthio-N6-isopentyladenine, uracil-5-oxyaceticacid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,methyl uracil-5-oxyacetate, uracil-5-oxyacetic acid (v), 5-methyl2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w and2,6-diaminopurine. Alternatively, the antisense nucleic acid can begenerated biologically using an expression vector to which a nucleicacid has been subcloned in antisense orientation (i.e. RNA which istranscribed by the nucleic acid introduced is in antisense orientationrelative to a target nucleic acid of interest, which is described ingreater detail in the subsection which follows).

The antisense nucleic acid molecules according to the invention areusually administered to a cell or generated in situ so that theyhybridize with, or bind to, the cellular mRNA and/or the genomic DNAencoding a PSE, thus inhibiting expression of the protein, for exampleby inhibiting transcription and/or translation. Hybridization can beeffected by conventional nucleotide complementarity with formation of astable duplex or, for example in the case of an antisense nucleic acidmolecule which binds DNA duplices, by specific interactions in the largecleft of the double helix. The antisense molecule can be modified insuch a manner that it specifically binds to a receptor or to an antigenexpressed at a selected cell surface, for example by binding theantisense nucleic acid molecule to a peptide or an antibody, each ofwhich binds to a cell surface receptor or an antigen. The cells can alsobe provided with the antisense nucleic acid molecule using the vectorsdescribed herein. Vector constructs in which the antisense nucleic acidmolecule is under the control of a strong prokaryotic, viral oreukaryotic promoter, inclusive of a plant promoter, are preferred forachieving sufficient intracellular concentrations of the antisensemolecules.

In a further embodiment, the antisense nucleic acid molecule accordingto the invention is an α-anomeric nucleic acid molecule. An α-anomericnucleic acid molecule forms specific double-stranded hybrids withcomplementary RNA, the strands running parallel to each other, incontrast to ordinary β-units [Gaultier et al. (1987) Nucleic Acids Res.15:6625-6641]. Moreover, the antisense nucleic acid molecule canencompass a 2′-o-methylribonucleotide [Inoue et al. (1987) Nucleic AcidsRes. 15:6131-6148] or a chimeric RNA DNA analogon [Inoue et al. (1987)FEBS Lett. 215:327-330].

In a further embodiment, an antisense nucleic acid according to theinvention is a ribozyme. Ribozymes are catalytic RNA molecules withribonuclease activity which can cleave a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes, for example hammerhead ribozymes [described in Haselhoff andGerlach (1988) Nature 334:585-591], can be used for the catalyticcleavage of PSE mRNA transcripts in order to inhibit the translation ofPSE mRNA. A ribozyme with specificity for a PSE-encoding nucleic acidcan be designed on the basis of the nucleotide sequence of a PSE cDNAdisclosed herein (i.e. 38° C.k21_g07fwd in SEQ ID NO:1, SEQ ID NO:3, SEQID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11) or on the basis of aheterologous sequence to be isolated in accordance with the processestaught in the present invention. For example, a derivative of aTetrahymena-L-19-IVS RNA can be constructed, in which the nucleotidesequence of the active site is complementary to the nucleotide sequenceto be cleaved in PSE-encoding mRNA. See, for example, Cech et al., U.S.Pat. No. 4,987,071 and Cech et al., U.S. Pat. No. 5,116,742. As analternative, PSE mRNA can be used for selecting a catalytic RNA with aspecific ribonuclease activity from amongst a pool of RNA molecules[see, for example, Bartel, D., and Szostak, J. W. (1993) Science261:1411-1418].

As an alternative, PSE gene expression can be inhibited by directingnucleotide sequences which are complementary to the regulatory region ofa PSE nucleotide sequence (for example a PSE promoter and/or enhancer)in such a way that triple helix structures are formed, which inhibit thetranscription of a PSE gene in target cells [see generally Helene, C.(1991) Anticancer Drug Res. 6(6) 569-84; Helene, C., et al. (1992) Ann.N.Y. Acad. Sci. 660:27-36; and Maher. L. J. (1992) Bioassays14(12):807-815].

B. Gene Construct

A further embodiment of the invention is a novel gene constructcomprising an isolated nucleic acid derived from Physcomitrella,Phytophthora, Crypthecodinium or Thraustochytrium and encodes apolypeptide which elongates C₁₆-, C₁₈- or C₂₀-fatty acids with at leasttwo double bonds in the fatty acid by at least two carbon atoms, orwhich comprises the gene sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11, its homologs,derivatives or analogs which are functionally linked to one or moreregulatory signals, advantageously for increasing gene expression.Examples of these regulatory sequences are sequences which bind toinductors or repressors, and in this manner regulate the expression ofthe nucleic acid. In addition to these novel regulatory sequences, thenatural regulation of these sequences before the actual structural genesmay still be present and, if appropriate, have been geneticallymodified, so that the natural regulation has been switched off and theexpression of the genes has been enhanced. However, the gene constructmay also have a simpler structure, i.e. no additional regulatory signalshave been inserted before the sequence SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11 or their homologs andthe natural promoter with its regulation has not been deleted. Instead,the natural regulatory sequence has been mutated in such a way thatregulation no longer takes place and gene expression is enhanced. Thegene construct may furthermore advantageously encompass one or moreso-called enhancer sequences which are functionally linked to thepromoter and which allow increased expression of the nucleic acidsequence. It is also possible additionally to insert advantageoussequences at the 3′ end of the DNA sequences, for example furtherregulatory elements or terminators. The elongase genes may be present inone or more copies in the gene construct. It is advantageous for theinsertion of further genes into organisms if further genes are presentin the gene construct.

Advantageous regulation sequences for the novel process exist, forexample, in promoters such as the cos, tac, trp, tet, trp-tet, lpp, lac,lpp-lac, lacIq, T7, T5, T3, gal, trc, ara, SP6, λ-P_(R) or λ-P_(L)promoter and are advantageously used in Gram-negative bacteria. Furtheradvantageous regulatory sequences exist, for example, in Grampositivepromoters amy and SPO2, in the yeast or fungal promoters ADC1, MFa, AC,P-60, CYCl, GAPDH, TEF, rp28, ADH or in the plant promoters CaMV/35S[Franck et al., Cell 21 (1980) 285-294], PRP1 [Ward et al., Plant. Mol.Biol. 22 (1993)], SSU, OCS, lib4, usp, STLS1, B33, nos or in theubiquitin or phaseolin promoter. Advantageous in this context are alsoinducible promoters, such as the promoters described in EP-A-0 388 186(benzylsulfonamide inducible), Plant J. 2, 1992:397-404 (Gatz et al.,tetracyclin-inducible), EP-A-0 335 528 (abscisic acid-inducible) or WO93/21334 (ethanol- or cyclohexenol-inducible). Other suitable plantpromoters are the cytosolic FBPase or the potato ST-LSI promoter(Stockhaus et al., EMBO J. 8, 1989, 2445), the Glycine maxphosphoribosylpyrophosphate amidotransferase promoter (Genbank AccessionNo. U87999) or the node-specific promoter described in EP-A-0 249 676.Especially advantageous promoters are promoters which allow expressionin tissues which are involved in fatty acid biosynthesis. Veryespecially advantageous are seed-specific promoters, such as the usp,the LEB4, the phaseolin or the napin promoter. Further especiallyadvantageous promoters are seed-specific promoters which can be used formonocots or dicots which are described in U.S. Pat. No. 5,608,152(oilseed rape napin promoter), WO 98/45461 (Arabidopsis phaseolinpromoter), U.S. Pat. No. 5,504,200 (Phaseolus vulgaris phaseolinpromoter), WO 91/13980 (Brassica Bce4-promoter), Baeumlein et al., PlantJ., 2, 2, 1992:233-239 (leguminous LEB4 promoter), these promoters beingsuitable for dicots. The following promoters are suitable, for example,for monocots: the barley lpt-2 or lpt-1 promoter (WO 95/15389 and WO95/23230), the barley hordein promoter, and other suitable promotersdescribed in WO 99/16890.

In principle, it is possible to use all natural promoters with theirregulatory sequences, such as those mentioned above, for the novelprocess. It is also possible and advantageous additionally to usesynthetic promoters.

As described above, the gene construct can also encompass further geneswhich are to be introduced into the organisms. It is possible andadvantageous to introduce into the host organisms, and to expresstherein, regulatory genes such as genes for inductors, repressors orenzymes which, owing to their enzymatic activity, engage in theregulation of one or more genes of a biosynthetic pathway. These genescan be of heterologous or homologous origin. The inserted genes can havetheir own promoter or else be under the control of the promoter ofsequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9and SEQ ID NO:11 or its homologs, derivatives or analogs.

To express the other genes which are present, the gene constructadvantageously encompasses further 3′- and/or 5′-terminal regulatorysequences for enhancing expression, and these are selected for optimalexpression as a function of the host organism chosen and the gene(s).

As mentioned above, these regulatory sequences are intended to makepossible the specific expression of the genes and protein expression.Depending on the host organism, this may mean, for example, that thegene is expressed or overexpressed only after induction, or that it isexpressed and/or overexpressed immediately.

Moreover, the regulatory sequences or regulatory factors can preferablyhave an advantageous effect on the expression of the genes which havebeen introduced, thus enhancing it. In this manner, it is possible thatthe regulatory elements are advantageously enhanced at thetranscriptional level, using strong transcription signals, such aspromoters and/or enhancers. However, it is furthermore also possible toenhance translation, for example by improving mRNA stability. Thenucleic acid sequences according to the invention are advantageouslycloned into a gene construct (=expression cassette, nucleic acidconstruct) together with at least one reporter gene, and this geneconstruct is introduced into the organism via a vector or directly intothe genome. This reporter gene should allow easy detectability by meansof a growth, fluorescence, chemoluminescence, bioluminescence orresistance assay or via photometric measurement. Examples of reportergenes which may be mentioned are genes for resistance to antibiotics orherbicides, hydrolase genes, fluorescence protein genes, bioluminescencegenes, sugar or nucleotide metabolism genes or biosynthesis genes suchas the Ura3 gene, the Ilv2 gene, the luciferase gene, theβ-galactosidase gene, the gfp gene, the 2-deoxyglucose-6-phosphatephosphatase gene, the β-glucuronidase gene, the β-lactamase gene, theneomycin phosphotransferase gene, the hygromycin phosphotransferase geneor the BASTA (=glufosinate resistance) gene. These genes make itpossible for the transcriptional activity, and thus gene expression, tobe measured and quantified readily. This allows the identification ofpositions in the genome which show different productivity.

The nucleic acid sequences according to the invention with SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11,which encode elongases, can be present in the expression cassette (=geneconstruct) in one or more copies.

The expression cassette (=gene construct, nucleic acid construct) canadditionally comprise at least one further nucleic acid which encodes agene, preferably from fatty acid biosynthesis, to be introduced into thehost organisms. These genes can be under separate regulation or underthe same regulatory region as the genes for the elongase according tothe invention. These genes are, for example, further biosynthesis genes,advantageously of fatty acid biosynthesis, which make possible anincreased synthesis. Genes which may be mentioned by way of example arethose for Δ19-, Δ17-, Δ15-, Δ12-, Δ9-, Δ8-, Δ6-, Δ5-, Δ4-desaturase, thevarious hydroxylases, Δ12-acetylenase, acyl-ACP thioesterases,β-ketoacyl-ACP synthases or β-ketoacyl-ACP reductases. The desaturasegenes are advantageously used in the nucleic acid construct. Again,these genes may be present in the gene construct in one or more copies.

C. Recombinant Expression Vectors and Host Cells

A further aspect of the invention relates to vectors, preferablyexpression vectors, comprising a nucleic acid according to the inventionor a gene construct according to the invention which encode a PSE (orpart thereof). As used in the present context, the term “vector” refersto a nucleic acid molecule which can transport another nucleic acid towhich it is bound. One type of vector is a “plasmid”, which represents acircular double-stranded DNA loop into which additional DNA segments canbe ligated. A further type of vector is a viral vector, it beingpossible for additional DNA segments to be ligated into the viralgenome. Certain vectors are capable of autonomous replication in a hostcell into which they have been introduced (for example bacterial vectorswith bacterial origin of replication and episomal mammalian vectors).Other vectors (for example nonepisomal mammalian vectors) are integratedinto the genome of a host cell upon introduction into the host cell andso replicate together with the host genome. In addition, certain vectorscan govern the expression of genes to which they are functionallylinked. These vectors are referred to as “expression vectors” herein.Usually, expression vectors which are suitable for recombinant DNAtechniques take the form of plasmids. In the present description,“plasmid” and “vector” may be used interchangeably since the plasmid isthe most frequently used form of a vector. However, the invention isintended to encompass these other forms of expression vectors, such asviral vectors (for example replication deficient retroviruses,adenoviruses and adeno-related viruses) which exert similar functions.Furthermore, the term vector is also intended to encompass other vectorsknown to the skilled worker, such as phages, viruses such as SV40, CMV,baculovirus, adenovirus, transposons, IS elements, phasmids, phagemids,cosmids, linear or circular DNA and RNA.

The recombinant expression vectors according to the invention encompassa nucleic acid according to the invention or a gene construct accordingto the invention in a form which is suitable for expressing the nucleicacid in a host cell, which means that the recombinant expression vectorsencompass one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is or are functionallylinked to the nucleic acid sequence to be expressed. In a recombinantexpression vector, “functionally linked” means that the nucleotidesequence of interest is bound to the regulatory sequence(s) in such away that expression of the nucleotide sequence is possible and they arebound to each other so that both sequences fulfill the predictedfunction which has been ascribed to the sequence (for example in anin-vitro transcription/translation system or in a host cell, when thevector is introduced into the host cell). The term “regulatory sequence”is intended to encompass promoters, enhancers and other expressioncontrol elements (for example polyadenylation signals). These regulatorysequences are described, for example, in Goeddel: Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990), or see: Gruber and Crosby, in: Methods in Plant MolecularBiology and Biotechnology, CRC Press, Boca Raton, Fla., Ed.: Glick andThompson, Chapter 7, 89-108, including the references therein.Regulatory sequences encompass those which control the constitutiveexpression of a nucleotide sequence in many types of host cell and thosewhich control the direct expression of the nucleotide sequence only incertain host cells under certain conditions. The skilled worker knowsthat the design of the expression vector may depend on factors such asthe choice of the host cell to be transformed, the extent to which thedesired protein is expressed, and the like. The expression vectorsaccording to the invention can be introduced into host cells in order toproduce proteins or peptides, including fusion proteins or fusionpeptides, which are encoded by the nucleic acids as described herein(for example PSEs, mutant forms of PSEs, fusion proteins and the like).

The recombinant expression vectors according to the invention can bedesigned for expressing PSEs in prokaryotic or eukaryotic cells. Forexample, PSE genes can be expressed in bacterial cells, such as C.glutamicum, insect cells (using baculovirus expression vectors), yeastand other fungal cells [see Romanos, M. A., et al. (1992) “Foreign geneexpression in yeast: a review”, Yeast 8:423-488; van den Hondel,C.A.M.J.J., et al. (1991) “Heterologous gene expression in filamentousfungi”, in: More Gene Manipulations in Fungi, J. W. Bennet & L. L.Lasure, Ed., pp. 396-428: Academic Press: San Diego; and van den Hondel,C.A.M.J.J., & Punt, P. J. (1991) “Gene transfer systems and vectordevelopment for filamentous fungi, in: Applied Molecular Genetics ofFungi, Peberdy, J. F., et al., Ed., pp. 1-28, Cambridge UniversityPress: Cambridge], algae [Falciatore et al., 1999, Marine Biotechnology.1, 3:239-251], ciliates of the following types: Holotrichia,Peritrichia, Spirotrichia, Suctoria, Tetrahymena, Paramecium, Colpidium,Glaucoma, Platyophrya, Potomacus, Pseudocohnilembus, Euplotes,Engelmaniella and Stylonychia, in particular of the genus Stylonychialemnae, using vectors and following a transformation method as describedin WO 98/01572, and cells of multicelled plants [see Schmidt, R. andWillmitzer, L. (1988) “High efficiency Agrobacteriumtumefaciens-mediated transformation of Arabidopsis thaliana leaf andcotyledon explants” Plant Cell Rep.:583-586; Plant Molecular Biology andBiotechnology, C Press, Boca Raton, Fla., Chapter 6/7, pp. 71-119(1993); F. F. White, B. Jenes et al., Techniques for Gene Transfer, in:Transgenic Plants, Bd. 1, Engineering and Utilization, Ed.: Kung and R.Wu, Academic Press (1993), 128-43; Potrykus, Annu. Rev. Plant Physiol.Plant Molec. Biol. 42 (1991), 205-225 (and references cited therein)] ormammalian cells. Suitable host cells are furthermore discussed inGoeddel, Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990). As an alternative, the recombinantexpression vector can be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.

In prokaryotes, proteins are usually expressed with vectors containingconstitutive or inducible promoters which control the expression offusion proteins or nonfusion proteins. Fusion vectors add a series ofamino acids to a protein encoded therein, usually at the amino terminusof the recombinant protein, but also at the C terminus or fused withinsuitable regions in the proteins. These fusion vectors usually havethree tasks: 1) to enhance the expression of recombinant protein; 2) toincrease the solubility of the recombinant protein and 3) to support thepurification of the recombinant protein by acting as ligand in affinitypurification, for example via so-called his tags. In the case of fusionexpression vectors, a proteolytic cleavage site is frequently introducedat the site where the fusion unit and the recombinant protein arelinked, so that the recombinant protein can be separated from the fusionunit after purification of the fusion protein. These enzymes and theircorresponding recognition sequences encompass factor Xa, thrombin andenterokinase.

Typical fusion expression vectors are, inter alia, pGEX [PharmaciaBiotech Inc; Smith, D. B., and Johnson, K. S. (1988) Gene 67:31-40],pMAL [New England Biolabs, Beverly, Mass.] and pRIT5 [Pharmacia,Piscataway, N.J.], where glutathione S-transferase (GST),maltose-E-binding protein or protein A is fused to the recombinanttarget protein. In one embodiment, the PSE-encoding sequence is clonedinto a pGEX expression vector to generate a vector encoding a fusionprotein which encompasses, from the N terminus to the C terminus,GST-thrombin cleavage site-X-protein. The fusion protein can be purifiedby affinity chromatography using glutathione-agarose resin. RecombinantPSE which is not fused with GST can be obtained by cleaving the fusionprotein with thrombin.

Examples of suitable inducible nonfusion E. coli expression vectors are,inter alia, pTrc (Amann et al. (1988) Gene 69:301-315) and pET 11d(Studier et al., Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 60-89). Target gene expressionof the pTrc vector is based on transcription by host RNA polymerase froma hybrid trp-lac fusion promoter. Target gene expression from the pET11d vector is based on transcription from a T7-gn10-lac fusion promoterwhich is mediated by a coexpressed viral RNA polymerase (T7 gn1). Thisviral polymerase is provided by the host strains BL21 (DE3) or HMS174(DE3) by a resident λ prophage which harbors a T7 gn1 gene under thetranscriptional control of the lacUV 5 promoter.

Other vectors which are suitable for use in prokaryotic organisms areknown to the skilled worker; these vectors are, for example, in E. colipLG338, pACYC184, the pBR series such as pBR322, the pUC series such aspUC18 or pUC19, the M113 mp series, pKC30, pRep4, pHS1, pHS2, pPLc236,pMBL24, pLG200, pUR290, pIN-III¹¹³-B1, pgt11 or pBdCI, in StreptomycespIJ101, pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194 or pBD214,in Corynebacterium pSA77 or pAJ667.

A strategy of maximizing the expression of recombinant protein is toexpress the protein in a host bacterium whose ability to cleave therecombinant protein proteolytically is disrupted [Gottesman, S., GeneExpression Technology Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128]. A further strategy is to modify thenucleic acid sequence of the nucleic acid to be inserted into anexpression vector, so that the individual codons for each amino acid arethose which are preferentially used in a bacterium selected forexpression, such as C. glutamicum [Wada et al. (1992) Nucleic Acids Res.20:2111-2118]. Modification of these nucleic acid sequences according tothe invention is carried out by standard techniques of DNA synthesis.

In a further embodiment, the PSE expression vector is a yeast expressionvector. Examples of vectors for expression in the yeast S. cerevisiaeinclude pYepSec1 [Baldari et al. (1987) Embo J. 6:229-234], pMFa [Kurjanand Herskowitz (1982) Cell 30:933-943], pJRY88 [Schultz et al. (1987)Gene 54:113-123] and pYES2 [Invitrogen Corporation, San Diego, Calif.].Vectors and methods for the construction of vectors which are suitablefor use in other fungi, such as the filamentous fungi, include thosewhich are described in detail in: van den Hondel, C.A.M.J.J., & Punt, P.J. (1991) “Gene transfer systems and vector development for filamentousfungi, in: Applied Molecular Genetics of fungi, J. F. Peberdy et al.,Ed., pp. 1-28, Cambridge University Press: Cambridge, or in: More GeneManipulations in Fungi [J. W. Bennet & L. L. Lasure, Ed., pp. 396-428:Academic Press: San Diego]. Further suitable-yeast vectors are, forexample, pAG-1, YEp6, YEp13 or pEMBLYe23.

As an alternative, the PSEs according to the invention can be expressedin insect cells using baculovirus expression vectors. Baculovirusvectors which are available for expressing proteins in cultured insectcells (for example Sf9 cells) include the pAc series [Smith et al.(1983) Mol. Cell Biol. 3:2156-2165] and the pVL series [Lucklow andSummers (1989) Virology 170:31-39].

The abovementioned vectors are just a short review of suitable vectorswhich are possible. Further plasmids are known to the skilled worker andare described, for example, in: Cloning Vectors (Ed. Pouwels, P. H., etal., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).

In yet a further embodiment, a nucleic acid according to the inventionis expressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 [Seed, B. (1987)Nature 329:840] and pMT2PC [Kaufman et al. (1987) EMBO J. 6:187-195].When used in mammalian cells, the control functions of the expressionvector are frequently provided by viral regulatory elements. Promoterswhich are usually used are derived, for example, from polyoma,adenovirus2, cytomegalovirus and simian virus 40. Other suitableexpression systems for prokaryotic and eukaryotic cells can be found inChapters 16 and 17 of Sambrook, J., Fritsch, E. F., and Maniatis, T.,Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector cancontrol the expression of the nucleic acid preferably in a specific celltype (for example, tissue-specific regulatory elements are used forexpressing the nucleic acid). Tissue-specific regulatory elements areknown in the art. Nonlimiting examples of suitable tissue-specificpromoters are, inter alia, the albumin promoter [liverspecific; Pinkertet al. (1987) Genes Dev. 1:268-277], lymphoid-specific promoters [Calameand Eaton (1988) Adv. Immunol. 43:235-275], in particular promoters ofT-cell receptors [Winoto and Baltimore (1989) EMBO J. 8:729-733] andimmunoglobulins [Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748], neuron-specific promoters [forexample neurofilament promoter; Byrne and Ruddle (1989) PNAS86:5473-5477], pancreas-specific promoters [Edlund et al., (1985)Science 230:912-916] and mamma-specific promoters [for example milkserum promoter; U.S. Pat. No. 4,873,316 and EP-A-0 264 166). Alsoincluded are development-regulated promoters, for example the mouse hoxpromoters [Kessel and Gruss (1990) Science 249:374-379] and thefetoprotein promoter [Campes and Tilghman (1989) Genes Dev. 3:537-546].

In a further embodiment, the PSEs according to the invention can beexpressed in single celled plant cells (such as algae), see Falciatoreet al., 1999, Marine Biotechnology 1 (3):239-251 and references citedtherein, and in plant cells from higher plants (for examplespermatophytes such as crops). Examples of plant expression vectorsinclude those which are described in detail in: Becker, D., Kemper, E.,Schell, J., and Masterson, R. (1992) “New plant binary vectors withselectable markers located proximal to the left border”, Plant Mol.Biol. 20:1195-1197; and Bevan, M. W. (1984) “Binary Agrobacteriumvectors for plant transformation”, Nucl. Acids Res. 12:8711-8721;Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Vol.1, Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press,1993, pp. 15-38. Further suitable plant vectors are described, interalia, in “Methods in Plant Molecular Biology and Biotechnology” (CRCPress), Chapter 6/7, pp. 71-119. Advantageous vectors are so-calledshuttle vectors or binary vectors, which replicate in E. coli andAgrobacterium.

A plant expression cassette preferably comprises regulatory sequenceswhich can control gene expression in plant cells and which arefunctionally linked, so that each sequence can fulfill its function,such as transcriptional termination, for example polyadenylationsignals. Preferred polyadenylation signals are those derived fromAgrobacterium tumefaciens T-DNA, such as gene 3 of the Ti plasmidpTiACH5, which is known as octopine synthase [Gielen et al., EMBO J. 3(1984) 835 et seq.] or functional equivalents thereof, but all otherterminators which are functionally active in plants are also suitable.

Since plant gene expression is very frequently not limited to thetranscriptional level, a plant expression cassette preferably comprisesother functionally linked sequences, such as translation enhancers, forexample the overdrive sequence, which contains the 5′-untranslatedtobacco mosaic virus leader sequence, which increases the protein/RNAratio [Gallie et al., 1987, Nucl. Acids Research 15:8693-8711].

Plant gene expression must be functionally linked to a suitable promoterwhich effects gene expression in a cell- or tissue-specific manner withthe correct timing. Preferred promoters are those which lead toconstitutive expression [Benfey et al., EMBO J. 8 (1989) 2195-2202],such as those which are derived from plant viruses such as 35S CAMV[Franck et al., Cell 21 (1980) 285-294], 19S CaMV (see also U.S. Pat.No. 5,352,605 and WO 84/02913) or plant promoters such as the Rubiscosmall subunit promoter described in U.S. Pat. No. 4,962,028.

Other sequences which are preferred for use for functional linkage inplant gene expression cassettes are targeting sequences, which arerequired for targeting the gene product in its corresponding cellcompartment [for a review, see Kermode, Crit. Rev. Plant Sci. 15, 4(1996) 285-423 and references cited therein], for example into thevacuole, the nucleus, all types of plastids such as amyloplasts,chloroplasts, chromoplasts, the extracellular space, the mitochondria,the endoplasmatic reticulum, elaioplasts, peroxisomes and othercompartments of plant cells.

Plant gene expression can also be facilitated via a chemically induciblepromoter [for a review, see Gatz 1997, Annu. Rev. Plant Physiol. PlantMol. Biol., 48:89-108]. Chemically inducible promoters are particularlysuitable when it is desired for gene expression to take place in aspecific manner with regard to timing. Examples of such promoters are asalicylic acid-inducible promoter (WO 95/19443), a tetracyclin-induciblepromoter [Gatz et al. (1992) Plant J. 2, 397-404] and anethanol-inducible promoter.

Other suitable promoters are promoters which respond to biotic orabiotic stress conditions, for example the pathogen-induced PRP1 genepromoter [Ward et al., Plant. Mol. Biol. 22 (1993) 361-366], theheat-inducible totomato hsp80 promoter (U.S. Pat. No. 5,187,267), thelow-temperature-inducible potato alpha amylase promoter (WO 96/12814) orthe wound-inducible pinII promoter (EP-A-0 375 091).

Promoters which are particularly preferred are those which lead to geneexpression in tissues and organs in which lipid and oil biosynthesistake place, in seed cells such as endosperm cells and cells of thedeveloping embryo. Promoters which are suitable are the oilseed rapenapin gene promoter (U.S. Pat. No. 5,608,152), the Vicia faba USPpromoter [Baeumlein et al., Mol Gen Genet, 1991, 225 (3):459-67], theArabidopsis oleosin promoter (WO 98/45461), the Phaseolus vulgarisphaseolin promoter (U.S. Pat. No. 5,504,200), the Brassica Bce4 promoter(WO 91/13980) or the legumin B4 promoter [LeB4; Baeumlein et al., 1992,Plant Journal, 2 (2):233-9], and promoters which lead to theseed-specific ex pression in monocots such as maize, barley, wheat, rye,rice and the like. Notable promoters which are suitable are the barleylpt2 or lpt1 gene promoter (WO 95/15389 and WO 95/23230), or thepromoters described in WO 99/16890 (promoters from the barley hordeingene, the rice glutelin gene, the rice oryzin gene, the rice prolamingene, the wheat gliadin gene, the wheat glutelin gene, the maize zeingene, the oat glutelin gene, the sorghum kasirin gene, and the ryesecalin gene).

Promoters which are also particularly suitable are those which lead toplastid-specific expression, since plastids are the compartment in whichthe precursors and some end products of lipid biosynthesis aresynthesized. Suitable promoters such as the viral RNA polymerasepromoter are described in WO 95/16783 and WO 97/06250, and theArabidopsis clpP promoter, described in WO 99/46394.

The invention furthermore provides a recombinant expression vectorencompassing a DNA molecule according to the invention which is clonedinto the expression vector in antisense orientation, i.e. the DNAmolecule is functionally linked to a regulatory sequence in such a waythat it allows the expression (by transcribing the DNA molecule) of anRNA molecule which is “antisense” to the PSE mRNA. Regulatory sequencesmay be selected which are functionally linked to a nucleic acid clonedin antisense orientation and which control the continuous expression ofthe antisense RNA molecule in a multiplicity of cell types, for example,viral promoters and/or enhancers or regulatory sequences may be selectedwhich control the constitutive, tissue-specific or cell type-specificexpression of antisense RNA. The antisense expression vector may bepresent in the form of a recombinant plasmid, phagemid or attenuatedvirus in which the antisense nucleic acids are produced under thecontrol of a highly effective regulatory region whose activity can bedetermined by the cell type into which the vector has been introduced.For an explanation of the regulation of gene expression by means ofantisense genes, see Weintraub, H., et al., Antisense-RNA as a moleculartool for genetic analysis, Reviews—Trends in Genetics, Vol. 1(1) 1986.

A further aspect of the invention relates to host cells into which arecombinant expression vector according to the invention has beenintroduced. The terms “host cell” and “recombinant host cell” are usedinterchangeably in the present context. Naturally, these terms do notonly refer to the particular target cell, but also to the progeny orpotential progeny of this cell. Since specific modifications may occurin subsequent generations owing to mutation or environmental effects,this progeny is not necessarily identical with the parental cell, butremains within the scope of the term as used in the present context.

A host cell may be a prokaryotic or eukaryotic cell. For example, a PSEcan be expressed in bacterial cells such as C. glutamicum, insect cells,fungal cells or mammalian cells (such as Chinese hamster ovary cells(CHO) or COS cells), algae, ciliates, plant cells, fungi or othermicroorganisms, such as C. glutamicum. Other suitable host cells areknown to the skilled worker.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. The terms“transformation” and “transfection”, conjugation and transduction asused in the present context are intended to encompass a multiplicity ofmethods known in the art for introducing foreign nucleic acid (forexample DNA) into a host cell, including calcium phosphate or calciumchloride coprecipitation, DEAE dextran mediated transfection,lipofection, natural competence, chemically mediated transfer,electroporation or particle bombardment. Suitable methods for thetransformation or transfection of host cells, including plant cells, canbe found in Sambrook et al. (Molecular Cloning: A Laboratory Manual.,2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989) and other laboratory textbooks,such as Methods in Molecular Biology, 1995, Vol. 44, Agrobacteriumprotocols, Ed.: Gartland and Davey, Humana Press, Totowa, N.J.

It is known about the stable transfection of mammalian cells that only aminority of the cells integrate the foreign DNA into their genome,depending on the expression vector used and the transfection techniqueused. To identify and select these integrants, a gene which encodes aselectable marker (for example resistance to antibiotics) is usuallyintroduced into the host cells together with the gene of interest.Preferred selectable markers encompass those which impart resistance todrugs such as G418, hygromycin and methotrexate, or, in plants, thosewhich impart resistance to a herbicide such as glyphosphate orglufosinate. Further suitable markers are, for example, markers whichencode genes which are involved in the biosynthesis pathways of, forexample, sugars or amino acids, such as β-galactosidase, ura3 or ilv2.Markers which encode genes such as luciferase, gfp or other fluorescencegenes are also suitable. These markers can be used in mutants in whichthese genes are not functional since they have been deleted for exampleby means of conventional methods. Furthermore, markers which encode anucleic acid which encodes a selectable marker can be introduced into ahost cell on the same vector as the one which encodes a PSE, or can beintroduced on a separate vector. Cells which have been transfectedstably with the nucleic acid introduced can be identified for example bydrug selection (for example, cells which have the selectable markerintegrated survive, whereupon other cells die).

To generate a homologously recombinant microorganism, a vector isgenerated which contains at least one segment of a PSE gene into which adeletion, addition or substitution has been introduced in order tomodify the PSE gene hereby, for example to functionally disrupt it. ThisPSE gene is preferably a Physcomitrella, Phytophthora, Crypthecodiniumor Thraustochytrium PSE gene, but a homolog or analog from otherorganisms, even from a mammalian, fungal or insect source, can also beused. In a preferred embodiment, the vector is designed in such a waythat the endogenous PSE gene is functionally disrupted (i.e. no longerencodes a functional protein, also termed knock-out vector) uponhomologous recombination. As an alternative, the vector can be designedsuch that the endogenous PSE gene mutates or is modified otherwise uponhomologous recombination while still encoding a functional protein (forexample, the upstream regulatory region can be modified in such a waythat this leads to a modification of the expression of the endogenousPSE). To generate a point mutation via homologous recombination, DNA-RNAhybrids, which are also known as chimeraplasty and which are known fromCole-Strauss et al., 1999, Nucleic Acids Research 27(5):1323-1330 andKmiec, Gene therapy, 1999, American Scientist, 87(3):240-247 can also beused.

In the vector for homologous recombination, the modified segment of thePSE gene is flanked at its 5′ and 3′ end by additional nucleic acid ofthe PSE gene, so that homologous recombination is possible between theexogenous PSE gene which is present on the vector and an endogenous PSEgene in a microorganism or a plant. The additional flanking PSE nucleicacid is sufficiently long for successful homologous recombination withthe endogenous gene. Usually, several hundred base pairs up to kilobasesof flanking DNA (both on the 5′ and on the 3′ end) are present in thevector [for a description of vectors for homologous recombination, see,for example, Thomas, K. R., and Capecchi, M. R. (1987) Cell 51:503 orfor the recombination in Physcomitrella patens on cDNA basis, see Streppet al., 1998, Proc. Natl. Acad. Sci. USA 95 (8):4368-4373]. The vectoris introduced into a microorganism or plant cell (for example by meansof polyethylene glycol-mediated DNA), and cells in which the PSE geneintroduced has undergone homologous recombination with the endogenousPSE gene are selected using techniques known in the art.

In another embodiment, recombinant organisms such as microorganisms ofthe plants can be generated which contain selected systems which allowregulated expression of the gene introduced. The inclusion of a PSE genein a vector, where it is placed under the control of the lac-operon,allows, for example, expression of the PSE gene only in the presence ofIPTG. These regulatory systems are known in the art.

A host cell according to the invention, such as prokaryotic oreukaryotic host cells, growing either in culture or in a field, can beused for producing (i.e. expressing) a PSE. In plants, an alternativemethod can additionally be used by directly transferring DNA intodeveloping flowers via electroporation or Agrobacterium mediated genetransfer. Accordingly, the invention furthermore provides methods ofproducing PSEs using the host cells according to the invention. In oneembodiment, the method encompasses growing the host cell according tothe invention (into which a recombinant expression vector encoding a PSEhas been introduced or into whose genome a gene encoding a wild-type ormodified PSE has been introduced) in a suitable medium until the PSE hasbeen produced. In a further embodiment, the method encompasses isolatingthe PSEs from the medium or the host cell.

Host cells which are suitable in principle for taking up the nucleicacid according to the invention, the novel gene product according to theinvention or the vector according to the invention are all prokaryoticor eukaryotic organisms. The host organisms which are usedadvantageously are organisms such as bacteria, fungi, yeasts, animalcells or plant cells. Further advantageous organisms are animals or,preferably, plants or parts thereof. The term “animal” is understoodhere as not including humans. Fungi, yeasts or plants are preferablyused, especially preferably fungi or plants, very especially preferablyplants such as oil crops which contain large amounts of lipid compounds,such as oilseed rape, evening primrose, castor oil plant, canola,peanut, linseed, soya, safflower, sunflower, borage, oil palm, coconutor plants such as maize, wheat, rye, oats, triticale, rice, barley,cotton, cassava, pepper, tagetes, Solanaceae plants such as potato,tobacco, aubergine and tomato, Vicia species, pea, alfalfa, shrubplants, (coffee, cacao, tea), Salix species, trees (oil palm, coconut)and perennial grasses and fodder crops. Especially preferred plantsaccording to the invention are oil crops such as soya, peanut, oilseedrape, canola, castor oil plant, linseed, evening primrose, sunflower,safflower, trees (oil palm, coconut).

A particularly preferred aspect of the invention relates to a plant cellwhich comprises the polynucleotide according to the invention or thevector according to the invention. Preference is furthermore given totransgenic plants or plant tissue comprising the plant cell according tothe invention. A further aspect of the present invention relates tothose parts of the plants according to the invention which can beharvested and to the material suitable for propagating the transgenicplants according to the invention, containing either plant cellsaccording to the invention which express the nucleic acid according tothe invention or containing cells which have an elevated level of theprotein according to the invention. In principle, all parts of a plantcan be harvested, in particular flowers, pollen, fruits, seedlings,roots, leaves, seeds, tubers, stems, etc. Propagation material includes,for example, seeds, fruits, seedlings, tubers, cuttings and rhizomes.

D. Isolated PSE

A further aspect of the invention relates to isolated PSEs andbiologically active parts thereof. An “isolated” or “purified” proteinor a biologically active part thereof, is essentially free of cellularmaterial when it is produced by recombinant DNA techniques, or free ofchemical precursors or other chemicals when it is synthesizedchemically. The term “essentially free of cellular material” encompassesPSE preparations in which the protein is separate from cellularcomponents of the cells in which it is produced naturally orrecombinantly. In one embodiment, the term “essentially free of cellularmaterial” encompasses PSE preparations with less than approximately 30%(based on the dry weight) of non-PSE (also referred to herein as“contaminating protein”), more preferably less than approximately 20% ofnon-PSE, even more preferably less than approximately 10% of non-PSE andmost preferably less than approximately 5% of non-PSE. When the PSE or abiologically active part thereof has been produced by recombinanttechnology, it is also essentially free of culture medium, i.e. theculture medium amounts to less than approximately 20%, more preferablyless than approximately 10% and most preferably less than approximately5% of the volume of the protein preparation. The term “essentially freeof chemical precursors or other chemicals” encompasses PSE preparationsin which the protein is separate from chemical precursors or otherchemicals which are involved in the synthesis of the protein. In oneembodiment, the term “essentially free of chemical precursors or otherchemicals” encompasses PSE preparations with less than approximately 30%(based on the dry weight) of chemical precursors or non-PSE chemicals,more preferably less than approximately 20% of chemical precursors ornon-PSE chemicals, even more preferably less than approximately 10% ofchemical precursors or non-PSE chemicals and most preferably less thanapproximately 5% of chemical precursors or non-PSE chemicals. Inpreferred embodiments, isolated proteins or biologically active partsthereof exhibit no contaminating proteins from the same organism fromwhich the PSE originates. In the case of the protein according to theinvention which contains the sequence shown in SEQ ID NO: 10 or which isencoded by a gene which comprises SEQ ID NO: 9, however, it has to betaken into account that the sequence starts with two Met. In thetranslation of a corresponding encoding nucleic acid sequence, this mayresult in the expression of two derivatives of the protein according tothe invention starting with the first or the second Met. The expressionratio between the two derivatives can vary between 0 and 1, depending onthe type of expression or the host organism. The invention accordinglycomprises PSE containing both of the derivatives mentioned, or only oneof the derivatives. The two derivatives can have different activities,localizations, half-lives, regulation mechanisms, etc. These proteinsare usually produced by recombinant expression, for examplePhyscomitrella, Phytophthora, Crypthecodinium or Thraustochytrium PSE inplants such as Physcomitrella patens or the abovementioned, ormicroorganisms for example bacteria such as E. coli, Bacillus subtilis,C. glutamicum, fungi such as Mortierella, yeast such as Saccharomyces,or ciliates such as Colpidium or algae such as Phaeodactylum.

An isolated PSE according to the invention or part thereof can alsoparticipate in the metabolism of compounds required for the synthesis ofcell membranes in Physcomitrella, Phytophthora, Crypthecodinium orThraustochytrium or in the transport of molecules via these membranes.In preferred embodiments, the protein or the part thereof encompasses anamino acid sequence which has sufficient homology with an amino acidsequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10 and SEQ ID NO:12 for the protein or the part thereof to retain theability to participate in the metabolism of compounds required for thesynthesis of cell membranes in Physcomitrella, Phytophthora,Crypthecodinium or Thraustochytrium or in the transport of molecules viathese membranes. The part of the protein is preferably a biologicallyactive part as described herein. In a further preferred embodiment, aPSE according to the invention has one of the amino acid sequences shownin SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 andSEQ ID NO:12. In a further preferred embodiment, the PSE has an aminoacid sequence which is encoded by a nucleotide sequence which hybridizeswith a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQID NO:7, SEQ ID NO:9 and SEQ ID NO:11, for example under stringentconditions. In yet another preferred embodiment, the PSE has an aminoacid sequence encoded by a nucleotide sequence which has at leastapproximately 50 to 60%, preferably at least approximately 60 to 70%,more preferably at least approximately 70 to 80%, 80 to 90%, 90 to 95%and more preferably at least approximately 96%, 97%, 98%, 99% or morehomology with one of the amino acid sequences of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12. The PSEpreferred in accordance with the invention preferably also has at leastone of the PSE activities described herein. For example, a preferred PSEaccording to the invention encompasses an amino acid sequence encoded bya nucleotide sequence which hybridizes with a nucleotide sequence of SEQID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ IDNO:11, for example under stringent conditions, and which can participatein the metabolism of compounds required for the synthesis of cellmembranes in Physcomitrella, Phytophthora, Crypthecodinium orThraustochytrium or in the transport of molecules via these membranesand is capable of elongating one or more polyunsaturated fatty acidswith at least two double bonds and a chain length of C₁₆ or C₁₈.

In other embodiments, the PSE is essentially homologous with an aminoacid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10 and SEQ ID NO:12 and retains the functional activity of theprotein of one of the sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12, their amino acidsequence differs, owing to natural variation or mutagenesis as describedin detail in the above subsection I. In a further embodiment, the PSEis, accordingly, a protein encompassing an amino acid sequence which hasat least approximately 50 to 60%, preferably at least approximately 60to 70% and more preferably at least approximately 70 to 80%, 80 to 90%,90 to 95% and most preferably at least approximately 96%, 97%, 98%, 99%or more homology with a complete amino acid sequence of SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO and SEQ ID NO:12 and has atleast one of the PSE activities described herein. In another embodiment,the invention relates to a complete Physcomitrella, Phytophthora,Crypthecodinium or Thraustochytrium protein which is essentiallyhomologous with a complete amino acid sequence of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12.

Biologically active parts of a PSE encompass peptides encompassing aminoacid sequences derived from the amino acid sequence of a PSE, forexample an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12 or the amino acidsequence of a protein which is homologous with a PSE, which peptideshave fewer amino acids than the full length PSE or the full-lengthprotein which is homologous with a PSE and have at least one activity ofa PSE. Biologically active parts (peptides, for example peptides with alength of, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50,100 or more amino acids) usually encompass a domain or a motif with atleast one activity of a PSE. Moreover, other biologically active partsin which other regions of the protein are deleted can be generated byrecombinant techniques and studied with regard to one or more of theactivities described herein. The biologically active parts of a PSEpreferably encompass one or more selected domains/motifs or partsthereof with biological activity.

Some of such domains and motifs can be identified by sequence analysis,for example using computer aided methods.

The sequences according to the invention were found to contain, forexample, KK motifs.

Kermode 1996, Critical Reviews in Plant Sciences 15 (4): 285-423,describes KK motifs, a double lysine, which is found mainly as KKXX or KX K XXX motif and which influences recycling from the ER to the Golgiapparatus and thus the residence time of the protein and its enzymeactivity at a certain location, in particular the ER.

Double lysine motifs have also been found, for example inΔ12-desaturases (Arondel et al. 1992, Science 258:1353), and they arealso present in the elongases according to the invention. In particularmotifs which can be localized at the C-terminus have been described. Inthe sequences according to the invention, there is a noticeableaccumulation of lysines at the C-terminus.

Moss elongase PSE1: C-terminus KQKGAKTE SEQ ID NO 4: C-terminus KTKKASEQ ID NO 6 C-terminus KKSTPAAKKTN SEQ ID NO 8: C-terminus KHLK

These may be a possible gene variation.

There are Lys radicals which influence targeting, addressing andlocalization at or in the ER. A masking of this sequence, modificationor spatial modification, in the vicinity of the end of the C-terminus,for example by fusion with GFP “green fluorescent protein” may beutilized to influence compartmentalization.

PSEs are preferably produced by recombinant DNA techniques. For example,a nucleic acid molecule encoding the protein is cloned into anexpression vector (as described above), the expression vector isintroduced into a host cell (as described above), and the PSE isexpressed in the host cell. The PSE can then be isolated from the cellsby a suitable purification scheme using standard techniques of proteinpurification. As an alternative for the recombinant expression, a PSE, aPSE polypeptide or a PSE peptide can be synthesized chemically bystandard techniques of peptide synthesis. Moreover, native PSE can beisolated from cells (for example endothelial cells), for example usingan anti-PSE antibody which can be raised by standard techniques, using aPSE according to the invention or a fragment thereof.

The invention also provides chimeric PSE proteins or PSE fusionproteins. As used in the present context, a “chimeric PSE protein” or“PSE fusion protein” encompasses a PSE polypeptide which is functionallybound to a non-PSE polypeptide. A “PSE polypeptide” refers to apolypeptide with an amino acid sequence which corresponds to a PSE,while a “non-PSE polypeptide” refers to a polypeptide with an amino acidsequence which corresponds to a protein which is essentially nothomologous with PSE, for example a protein which differs from PSE andwhich originates from the same or another organism. Within the fusionprotein, the term “functionally linked” is to be understood as meaningthat the PSE polypeptide and the non-PSE polypeptide are fused to eachother in such a way that both sequences fulfill the predicted functionwhich has been ascribed to the sequence used. The non-PSE polypeptidecan be fused to the N terminus or the C terminus of the PSE polypeptide.In one embodiment the fusion protein is, for example, a GST-PSE fusionprotein in which the PSE sequences are fused to the C terminus of theGST sequences. These fusion proteins can facilitate the purification ofthe recombinant PSEs. In a further embodiment, the fusion protein is aPSE which has a heterologous signal sequence at its N terminus. Incertain host cells (for example mammalian host cells), expression and/orsecretion of a PSE can be increased by using a heterologous signalsequence.

A chimeric PSE protein or PSE fusion protein according to the inventionis produced by standard recombinant DNA techniques. For example, DNAfragments which encode different polypeptide sequences are ligated toeach other in correct reading frame using conventional techniques, forexample by employing blunt ends or sticky ends for ligation, restrictionenzyme cleavage for providing suitable ends, filling up cohesive ends,as required, treatment with alkaline phosphatase to avoid undesiredlinkages, and enzymatic ligation. In a further embodiment, the fusiongene can be synthesized by conventional techniques including DNAsynthesizers. As an alternative, PCR amplification of gene fragments canbe carried out using anchor primers which generate complementaryoverhangs between successive gene fragments which can subsequently behybridized and reamplified to give rise to a chimeric gene sequence(see, for example, Current Protocols in Molecular Biology, Ed. Ausubelet al., John Wiley & Sons: 1992). Moreover, a large number of expressionvectors which already encode a fusion unit (for example a GSTpolypeptide) are commercially available. PSE-encoding nucleic acid canbe cloned into such an expression vector so that the fusion unit islinked in correct reading frame to the PSE protein.

PSE homologs can be generated by mutagenesis, for example by specificpoint mutation or truncating the PSE. The term “homologs” as used in thepresent context refers to a variant form of PSE which acts as agonist orantagonist of the PSE activity. A PSE agonist can essentially retain thesame activity as PSE, or some of the biological activities. A PSEantagonist can inhibit one or more activities of the naturally occurringPSE form, for example by competitive binding to an upstream ordownstream element of the metabolic cascade for cell membrane componentswhich encompasses the PSE, or by binding to a PSE which mediates thetransport of compounds via cell membranes, thus inhibitingtranslocation.

In an alternative embodiment, PSE homologs can be identified byscreening combinatory libraries of mutants, for example truncatedmutants, of PSE with regard to PSE agonist or PSE antagonist activity.In one embodiment, a variegated library of PSE variants is generated atthe nucleic acid level by combinatory mutagenesis and encoded by avariegated genetic library. A variegated library of PSE variants can begenerated for example by enzymatic ligation of a mixture of syntheticoligonucleotides into gene sequences so that a degenerate set ofpotential PSE sequences can be expressed as individual polypeptides or,alternatively, as a set of larger fusion proteins (for example for phagedisplay) which comprise this set of PSE sequences. There is amultiplicity of methods which can be used for generating libraries ofpotential PSE homologs from a degenerate oligonucleotide sequence. Thechemical synthesis of a degenerate gene sequence can be carried out in aDNA synthesizer, and the synthetic gene can then be ligated into asuitable expression vector. The use of a degenerate set of genes allowsall sequences which encode the desired set of potential PSE sequences tobe provided in a mixture. Methods for the synthesis of degenerateoligonucleotides are known in the art [see, for example, Narang, S. A.(1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem.53:323; Itakura et al., (1984) Science 198:1056; Ike et al. (1983)Nucleic Acids Res. 11:477].

In addition, libraries of PSE fragments can be used for generating avariegated population of PSE fragments for screening and for thesubsequent selection of homologs of a PSE. In one embodiment, a libraryof fragments of the coding sequence can be generated by treating adouble-stranded PCR fragment of a coding PSE sequence with a nucleaseunder conditions under which double-strand breaks only occurapproximately once per molecule, denaturing the double-stranded DNA,renaturing the DNA with the formation of double-stranded DNA which canencompass sense/antisense pairs of various products with double-strandbreaks, removal of single-stranded sections from newly formed duplicesby treatment with S1 nuclease, and ligating the resulting fragmentlibrary into an expression vector. This method allows an expressionlibrary to be derived which encodes N-terminal, C-terminal and internalfragments of variously sized PSEs.

A number of techniques for screening gene products in combinatorylibraries which have been generated by point mutations or truncation andfor screening cDNA libraries for gene products with a selected propertyare known in the art. These techniques can be adapted to rapid screeningof the genetic libraries which have been generated by combinatorymutagenesis of PSE homologs. The most frequently used techniques forscreening large genetic libraries which can be subjected tohigh-throughput analysis usually encompass cloning the genetic libraryinto replicable expression vectors, transforming suitable cells with theresulting vector library, and expressing the combinatory genes underconditions under which detecting the desired activity facilitates theisolation of the vector encoding the gene whose product has beendetected. Recursive ensemble mutagenese (REM), a novel technique whichincreases the frequency of functional mutants in the libraries, can beused in combination with the screening assays for identifying PSEhomologs [Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331].Combinations of the abovementioned methods can also be usedadvantageously.

A further known technique for modifying catalytic properties of enzymesor the genes encoding them is gene shuffling (see, for example, WO97/20078 or WO 98/13487), which is a combination of gene fragments wherethis new combination can additionally be varied by erroneous polymerasechain reactions thus creating a high sequence diversity to be assayed.However, the prerequisite for using such an approach is a suitablescreening system to test the resulting gene diversity for functionality.

A screening method which identifies a PUFA-dependent enzyme activity, oractivities, is a prerequisite in particular for screening elongaseactivities. As regards elongase activities with a specificity for PUFAs,the toxicity of arachidonic acid in the presence of a toxic metabolite(here: salicylic acid or salicylic acid derivatives), can be exploitedin Mucor species which can be transformed with desired gene constructsby known transformation methods (Eroshin et al., Mikrobiologiya, Vol.65, No. 1, 1996, pages 31-36) to carry out a growth based primaryscreening. Resulting clones can then be analyzed for their lipidconstituents by means of gas chromatography and mass spectroscopy inorder to identify the nature and quantity of starting materials andproducts.

In a further embodiment, cell-based assays can be made use of foranalyzing a variegated PSE library using further processes known in theart.

In a further embodiment, the present invention relates to an antibodywhich binds specifically to the polypeptide of the present invention orto parts, for example epitopes, of such a protein. The antibodyaccording to the invention can be used to identify and isolate otherelongases, in particular PSEs. These antibodies can be monoclonalantibodies, polyclonal antibodies or synthetic antibodies, and alsofragments of these antibodies, such as, for example, Fab, Fv or scFVfragments, etc. Monoclonal antibodies can be prepared, for example, bymethods such as those described originally by Köhler and Milstein inNature 256 (1975), 485, and Galfrö in Meth. Enzymol. 73 (1981).Antibodies and fragments thereof can also be prepared, for example,according to Harlow & Lane, “Antibodies, a Laboratory Manual”, CSHPress, Cold Spring Harbor, 1988. These antibodies can be used toprecipitate and localize, for example, the proteins according to theinvention, or to monitor the synthesis of these proteins, for example inrecombinant organisms, and to identify compounds which interact with theproteins according to the invention. In many cases, the binding ofantibodies to antigens is equivalent to the binding of other ligands andantiligands.

The present invention furthermore relates to a process for identifyingan agonist or antagonist of elongases, in particular PSEs, comprising

-   a) bringing the cells which express the polypeptide of the present    invention into contact with a candidate substance;-   b) testing the PSE activity;-   c) comparing the PSE activity with a standard activity in the    absence of the candidate substance, where a PSE activity that is    higher than that of the standard indicates that the candidate    substance is an agonist and where a PSE activity that is lower than    that of the standard indicates that the candidate substance is an    antagonist.

The candidate substance mentioned can be a substance that is synthesizedchemically or produced microbiologically, being present, for example, incell extracts of, for example, plants, animals or microorganisms. Thesubstance mentioned may furthermore be known in the prior art buthitherto unknown as increasing or repressing the activity of the PSEs.The reaction mixture may be a cell free extract or comprise a cell orcell culture. Suitable methods are known to the person skilled in theart and described in a general manner, for example, in Alberts,Molecular Biology of the cell, 3^(rd) edition (1994), for exampleChapter 17. The substances mentioned may be added, for example, to thereaction mixture or the culture medium or be injected into the cells orsprayed onto a plant.

If a sample which comprises a substance which is active according to themethod according to the invention has been identified, it is thenpossible either to isolate the substance directly from the originalsample or to divide the sample into various groups, for example when itconsists of a large number of different components, to reduce the numberof different substances per sample, and then to repeat the processaccording to the invention with such a “subsample” of the originalsample. Depending on the complexity of the sample, the steps describedabove can be repeated a plurality of times, preferably until the sampleidentified by the method according to the invention comprises only asmall number of substances or only one substance. The substanceidentified by the method according to the invention, or derivativesthereof, are preferably formulated further, such that they are suitablefor use in plant breeding or plant cells or tissue culture.

Substances which can be identified and tested by the process accordingto the invention can be: expression libraries, for example cDNAexpression libraries, peptides, proteins, nucleic acids, antibodies,small organic substances, hormones, PNAs or the like (Milner, NatureMedicin 1 (1995), 879-880; Hupp, Cell. 83 (1995), 237-245; Gibbs, Cell.79 (1994), 193-198 and literature quoted therein). These substances canalso be functional derivatives or analogs of known inhibitors oractivators. Processes for preparing chemical derivatives or analogs areknown to the person skilled in the art. The known derivatives andanalogs can be tested using processes of the prior art. It isfurthermore possible to use computer-aided design or peptidomimetics toprepare suitable derivatives and analogs. The cell or the tissue usedfor the process(es) according to the invention is preferably a hostcell, plant cell or a plant tissue according to the invention, asdescribed in the embodiments above.

Correspondingly, the present invention also relates to a substance whichwas identified by the process according to the invention mentionedabove. The substance is, for example, a homolog of the PSE according tothe invention. Homologs of the PSEs can be generated by mutagenesis, forexample by point mutation or deletion of PSE. Here, the term “homolog”is used to mean a variant form of the PSEs which acts as agonist orantagonist for the PSE activity. An agonist may have substantially thesame or part of the biological activity of the PSEs. An antagonist ofthe PSEs may inhibit one or more activities of the naturally occurringforms of the PSEs, for example bind competitively to a downstream orupstream member of the metabolic paths of fatty acid synthesis includingthe PSEs, or bind to PSEs and reducing or inhibiting the activity in theprocess.

Accordingly, the present invention also relates to an antibody or afragment thereof as described herein which inhibits the activity of thePSEs according to the invention.

One aspect of the present invention relates to an antibody whichspecifically recognizes or binds to the above-described agonist orantagonist according to the invention.

A further aspect relates to a composition which comprises the anti body,the stop or the antisense molecule identified by the process accordingto the invention.

E. Uses and Processes/Methods According to the Invention

The nucleic acid molecules, proteins, protein homologs, fusion proteins,antibodies, primers, vectors and host cells described herein can be usedin one or more of the methods which follow: identification ofPhyscomitrella patens, Crypthecodinium, Phytophthora infestans orThraustochytrium and related organisms, genome mapping of organismswhich are related with Physcomitrella, Phytophthora, Crypthecodinium orThraustochytrium, identification and localization of Physcomitrella,Phytophthora, Crypthecodinium or Thraustochytrium sequences of interest,evolutionary studies, determination of PSE protein regions required forthe function, modulation of a PSE activity; modulation of the metabolismof one or more cell membrane components; modulation of the transmembranetransport of one or more compounds, and modulation of the cellularproduction of a desired compound such as a fine chemical. The PSEnucleic acid molecules according to the invention have a multiplicity ofuses. Firstly, they can be used for identifying an organism asPhyscomitrella, Phytophthora, Crypthecodinium or Thraustochytrium or aclose relative of these. They can also be used for identifying thepresence of Physcomitrella, Crypthecodinium, Phytophthora orThraustochytrium or a relative of these in a mixed population ofmicroorganisms. The invention provides the nucleic acid sequences of aseries of Physcomitrella, Phytophthora, Crypthecodinium orThraustochytrium genes; the presence or absence of this organism can bedetermined by screening the extracted genomic DNA of a culture of auniform or mixed population of microorganisms under stringent conditionswith a probe covering a region of a Physcomitrella, Crypthecodinium,Phytophthora or Thraustochytrium gene which is unique for this organism,or of parts of this gene. Physcomitrella, Crypthecodinium, Phytophthoraor Thraustochytrium themselves are used for the commercial production ofpolyunsaturated acids. Moreover, the nucleic acids according to theinvention are suitable for the production of PUFAs, also in otherorganisms, in particular when it is intended for the resulting PUFAs tobe incorporated into the triacylglycerol fraction.

Furthermore, the nucleic acid and protein molecules according to theinvention can act as marker for specific regions of the genome. They arenot only suitable for mapping the genome, but also for functionalstudies of Physcomitrella, Phytophthora, Crypthecodinium orThraustochytrium proteins. To identify the genome region to which acertain DNA-binding protein of Physcomitrella, Crypthecodinium,Phytophthora or Thraustochytrium binds, the Physcomitrella,Crypthecodinium, Phytophthora or Thraustochytrium genome can befragmented, for example, and the fragments incubated with theDNA-binding protein. Those which bind the protein can additionally bescreened with the nucleic acid molecules according to the invention,preferably with readily detectable markers; the binding of such anucleic acid molecule to the genome fragment makes possible thelocalization of the fragment on the genome map of Physcomitrella,Phytophthora, Crypthecodinium or Thraustochytrium and, if this iscarried out repeatedly with different enzymes, facilitates a rapiddetermination of the nucleic acid sequence to which the protein binds.Moreover, the nucleic acid molecules according to the invention can havesufficient homology with the sequences of related species for thesenucleic acid molecules to be able to act as markers for the constructionof a genomic map in related fungi or algae.

The PSE nucleic acid molecules according to the invention are alsosuitable for evolutionary studies and studies of the protein structure.The metabolic and transport processes in which the molecules accordingto the invention are involved are utilized by a large number ofprokaryotic and eukaryotic cells; the evolutionary degree of relatednessof the organisms can be determined by comparing the sequences of thenucleic acid molecules according to the invention which those whichencode similar enzymes from other organisms. Accordingly, such acomparison allows the determination of which sequence regions areconserved and which are not conserved, which may be helpful whendetermining regions of the protein which are essential for enzymefunction. This type of determination is valuable for protein engineeringstudies and may provide a clue of how much mutagenesis the protein cantolerate without losing its function.

Manipulation of the PSE nucleic acid molecules according to theinvention can lead to the production of PSEs with functional differencesto the wild-type PSEs. The efficacy or activity of these proteins can beimproved; they may be present in the cell in larger numbers than usual;or their efficacy or activity can be reduced. An improved efficacy oractivity means, for example, that the enzyme has a higher selectivityand/or activity, preferably an activity which is at least 10% higher,very especially an activity which is at least 20% higher, veryespecially preferably an activity which is at least 30% higher, than theoriginal enzyme.

There exists a series of mechanisms by which modification of a PSEaccording to the invention can directly affect yield, production and/orproduction efficacy of a fine chemical comprising such a modifiedprotein. Obtaining fine chemical compounds from cultures of ciliates,algae or fungi on a large scale is significantly improved when the cellsecretes the desired compounds, since these compounds can readily beisolated from the culture medium (in contrast to extraction from thebiomass of the cultured cells). Otherwise, purification can be improvedwhen the cell stores compounds in vivo in a specialized compartment witha sort of concentration mechanism. In plants which express PSEs, anincreased transport may lead to better distribution within the planttissue and the plant organs. Increasing the number or the activity ofthe transporter molecules which export fine chemicals from the cell mayallow the quantity of the fine chemical produced, which is present inthe extracellular medium, to be increased, thus facilitating harvestingand purification or, in the case of plants, more efficient distribution.In contrast, increased amounts of cofactors, precursor molecules andintermediates for the suitable biosynthetic pathways are required for anefficient overproduction of one or more fine chemicals. Increasing thenumber and/or the activity of transporter proteins involved in theimport of nutrients such as carbon sources (i.e. sugars), nitrogensources (i.e. amino acids, ammonium salts), phosphate and sulfur canimprove the production of a fine chemical owing to the elimination ofall limitations of the nutrients available in the biosynthetic process.Fatty acids such as PUFAs and lipids comprising PUFAs are desirable finechemicals themselves. Optimizing the activity or increasing the numberof one or more PSEs according to the invention involved in thebiosynthesis of these compounds, or disrupting the activity of one ormore PSEs involved in the breakdown of these compounds, can thusincrease the yield, production and/or production efficacy of fatty acidand lipid molecules in ciliates, algae, plants, fungi, yeasts or othermicroorganisms.

The manipulation of one or more PSE genes according to the invention canlikewise lead to PSEs with modified activities which indirectly affectthe production of one or more desired fine chemicals from algae, plants,ciliates or fungi. The normal biochemical metabolic processes lead, forexample, to the production of a multiplicity of waste products (forexample hydrogen peroxide and other reactive oxygen species) which canactively disrupt these metabolic processes [for example, peroxynitriteis known to nitrate tyrosin side chains, thus inactivating some enzymeswith tyrosin in the active center; Groves, J. T. (1999) Curr. Opin.Chem. Biol. 3(2); 226-235]. While these waste products are normallyexcreted, the cells used for fermentative production on a large scaleare optimized for the overproduction of one or more fine chemicals andcan therefore produce more waste products than is customary for awild-type cell. Optimizing the activity of one or more PSEs according tothe invention which are involved in the export of waste molecules allowsthe improvement of the viability of the cell and the maintenance of anefficient metabolic activity. Also, the presence of high intracellularamounts of the desired fine chemical can in fact be toxic to the cell,so that the viability of the cell can be improved by increasing theability of the cell to secrete these compounds.

Furthermore, the PSEs according to the invention can be manipulated insuch a way that the relative amounts of various lipid and fatty acidmolecules are modified. This can have a decisive effect on the lipidcomposition of the cell membrane. Since each lipid type has differentphysical properties, the modification of the lipid composition of amembrane can significantly modify membrane fluidity. Changes in membranefluidity can affect the transport of molecules via the membrane which,as explained above, can modify the export of waste products or of thefine chemical produced or the import of nutrients which are required.These changes in membrane fluidity can also have a decisive effect oncell integrity; cells with comparatively weaker membranes are moresusceptible to abiotic and biotic stress conditions which can damage orkill the cell. Manipulation of PSEs which are involved in the productionof fatty acids and lipids for membrane synthesis so that the resultingmembrane has a membrane composition which is more susceptible to theenvironmental conditions prevailing in the cultures used for theproduction of fine chemicals should allow more cells to survive andmultiply. Larger numbers of producing cells should manifest themselvesin greater yields, higher production or higher production efficacy ofthe fine chemical from the culture.

The abovementioned mutagenesis strategies for PSEs intended to lead toelevated yields of a fine chemical are not to be construed as limiting;variations of these strategies are readily obvious to the skilledworker. Using these mechanisms, and with the aid of the mechanismsdisclosed herein, the nucleic acid and protein molecules according tothe invention can be used for generating algae, ciliates, plants,animals, fungi or other microorganisms such as C. glutamicum whichexpress mutated PSE nucleic acid and protein molecules so that theyield, production and/or production efficacy of a desired compound isimproved. This desired compound can be any natural product of algae,ciliates, plants, animals, fungi or bacteria which encompasses the endproducts of biosynthetic pathways and intermediates of naturallyoccurring metabolic pathways, and also molecules which do not naturallyoccur in the metabolism of these cells, but which are produced by thecells according to the invention.

A further embodiment according to the invention is a process for theproduction of PUFAs, which comprises culturing an organism whichcontains a nucleic acid according to the invention, a gene constructaccording to the invention or a vector according to the invention whichencode a polypeptide which elongates C₁₆-, C₁₈- or C₂₀-fatty acids withat least two double bonds in the fatty acid molecule by at least twocarbon atoms under conditions under which PUFAs are produced in theorganism. PUFAs prepared by this process can be isolated by harvestingthe organisms either from the culture in which they grow or from thefield, and disrupting and/or extracting the harvested material with anorganic solvent. The oil, which contains lipids, phospholipids,sphingolipids, glycolipids, triacylglycerols and/or free fatty acidswith a higher PUFA content, can be isolated from this solvent. The freefatty acids with a higher content of PUFAs can be isolated by basic oracid hydrolysis of the lipids, phospholipids, sphingolipids, glycolipidsand triacylglycerols. A higher content of PUFAS means at least 5%,preferably 10%, especially preferably 20%, very especially preferably40% more PUFAs than the original organism which does not have additionalnucleic acid encoding the elongase according to the invention.

The PUFAs produced by this process are preferably C₁₈-, C₂₀- orC₂₂-fatty acid molecules with at least two double bonds in the fattyacid molecule, preferably three, four, five or six double bonds,especially preferably three or five double bonds. These C₁₈-, C₂₀- orC₂₂-fatty acid molecules can be isolated from the organism in the formof an oil, lipid or a free fatty acid. Examples of suitable organismsare those mentioned above. Preferred organisms are microorganisms,animals or plants, especially preferably plants or algae, veryespecially preferably transgenic plants.

An embodiment according to the invention are oils, lipids or fatty acidsor fractions thereof which have been prepared by the above-describedprocess, especially preferably oil, lipid or a fatty acid compositionencompassing PUFAs and originating from transgenic plants.

One embodiment of the invention are oils, lipids or fatty acids whichhave been prepared by the process according to the invention. Otherembodiments of the invention are oil, lipid or fatty acid compositionswhich comprise PUFAs produced by the process according to the inventionand which are derived from transgenic plants which comprise a nucleicacid, a gene construct or vector according to the invention.

A further embodiment according to the invention is the use of the oil,lipid or the fatty acid composition in feeding stuffs, foodstuffs,cosmetics or pharmaceuticals.

A further embodiment of the invention relates to a kit, comprising thenucleic acid according to the invention, the gene construct according tothe invention, the amino acid sequence according to the invention, theantisense nucleic acid molecule according to the invention, the antibodyand/or composition according to the invention, an antagonist or agonistprepared by the process according to the invention and/or oils, lipidsand/or fatty acids according to the invention, or a fraction thereof.The kit may also comprise the host cells, organisms or plants accordingto the invention, or parts thereof, parts of the plants according to theinvention which can be harvested, or propagation material, or else theantagonist or agonist according to the invention. The components of thekit of the present invention can be packed in suitable containers, forexample together with or in buffers or other solutions. One or more ofthe components mentioned may be packed into one and the same container.Additionally or alternatively, one or more of the components mentionedcan, for example, be absorbed on a solid surface, for example anitrocellulose filter, glass plates, chips, nylon membranes ormicrotiter plates. The kit can be used for any of the methods andembodiments described herein, for example for producing host cells,transgenic plants, for detecting homologous sequences, for identifyingantagonists or agonists, and the like. Furthermore, the kit may containinstructions on how to use the kit for one of the applicationsmentioned.

This invention is illustrated in greater detail by the examples whichfollow, which are not to be construed as limiting. The content of allreferences, patent applications, patents and published patentapplications cited in this patent application is incorporated herein byreference.

EXAMPLES Example 1 General Methods

a) General Cloning Methods:

Cloning methods, such as, for example, restriction cleavages, agarosegel electrophoresis, purification of DNA fragments, transfer of nucleicacids to nitrocellulose and nylon membranes, linkage of DNA fragments,transformation of Escherichia coli and yeast cells, the culture ofbacteria and the sequence analysis of recombinant DNA were carried outas described in Sambrook et al. [(1989), Cold Spring Harbor LaboratoryPress: ISBN 0-87969-309-6] or Kaiser, Michaelis and Mitchell [(1994),“Methods in Yeast Genetics”, Cold Spring Harbor Laboratory Press: ISBN0-87969-451-3]. The transformation and culture of algae such asChlorella or Phaeodactylum are carried out as described by El-Sheekh[(1999), Biologia Plantarum 42:209-216] or Apt et al. [(1996) Molecularand General Genetics 252 (5):872-9].

b) Chemicals

Unless otherwise specified in the text, the chemicals used were obtainedin analytical grade quality from Fluka (Neu-Ulm), Merck (Darmstadt),Roth (Karlsruhe), Serva (Heidelberg) and Sigma (Deisenhofen). Solutionswere prepared using pure pyrogen free water, referred to in thefollowing text as H₂O, from a Milli-Q water system water purificationunit (Millipore, Eschborn). Restriction endonucleases, DNA-modifyingenzymes and molecular biology kits were obtained from AGS (Heidelberg),Amersham (Brunswick), Biometra (Gottingen), Boehringer (Mannheim),Genomed (Bad Oeynhausen), New England Biolabs (Schwalbach/Taunus),Novagen (Madison, Wis., USA), Perkin-Elmer (Weiterstadt), Pharmacia(Freiburg), Qiagen (Hilden) and Stratagene (Amsterdam, Netherlands).Unless otherwise specified, they were used following the manufacturer'sinstructions.

c) Cell Material

The present studies were carried out using a Thraustochytrium strainwhich is available via the American Type Culture Collection (ATCC) withthe strain number ATCC26185 (Thraustochytrium) or, in the case ofCrypthecodinium, from the Provasoli-Guillard National Center for Cultureof Marine Phytoplankton ((CCMP) West Boothbay Harbour, Me., USA), withthe strain culture No. CCMP316. The algae were cultured in the dark at25 degrees Celsius in glass tubes into which air was passed in from thebottom. As an alternative, Thraustochytrium was grown as described indetail by Singh & Ward (1997, Advances in Microbiology, 45, 271-311).

The culture medium used for Crypthecodinium was the f/2 culture mediumsupplemented with 10% organic medium of Guillard, R. R. L. [1975;Culture of phytoplankton for feeding marine invertebrates. In: Smith, W.L. and Chanley, M. H. (Eds.) Culture of marine Invertebrate animals, NYPlenum Press, pp. 29-60.]. It comprises

995.5 ml of (artificial) salt water

1 ml NaNO₃ (75 g/l), 1 ml NaH₂PO₄ (5 g/l), 1 ml trace metal solution, 1ml Tris/Cl pH 8.0, 0.5 ml f/2 vitamin solution

Trace metal solution: Na₂EDTA (4.36 g/l), FeCl₃ (3.15 g/l),

Primary Trace metals: CuSO₄ (10 g/l), ZnSO₄ (22 g/l), CoCl₂ (10 g/l),MnCl₂ (18 g/l), NaMoO₄ (6.3 g/l)

f/2 vitamin solution: biotin: 10 mg/l, thiamin 200 mg/l, vitamin B12 0.1mg/l

org medium: sodium acetate (1 g/l), glucose (6 g/l), sodium succinate (3g/l), Bacto tryptone (4 g/l), yeast extract (2 g/l)

d) Moss Material (=Plant Material)

For this study, plants of the species Physcomitrella patens (Hedw.)B.S.G. from the collection of the department for genetic studies,University of Hamburg, were used. They are derived from strain 16/14,which had been collected by H.L.K. Whitehouse in Gransden Wood,Huntingdonshire (England) and subcultured by Engel (1968, Am J Bot 55,438-446) from a spore. Proliferation of the plants was done by means ofspores and by regenerating the gametophytes. The protonema developedfrom the haploid spore into chloroplast-rich chloronema andchloroplast-depleted caulonema, which budded after approximately 12days. These buds grew into gametophores with antheridia and archegonia.Fertilization gave rise to diploid sporophyte with short seta and sporecapsule in which the meiospores mature.

e) Plant Culture

Plants were grown in a controlled environment cabinet at an airtemperature of 25° C. and a light intensity of 55 μmol⁻¹ m⁻² (whitelight; Philips TL 65W/25 fluorescent tube) and a light/dark region of16/8 hours. The moss was grown either in liquid culture using Reski andAbel's modified knop medium (1985, Planta 165, 354-358) or on solid knopmedium using 1% Oxoid agar (Unipath, Basingstoke, England).

The protonemata used for RNA and DNA isolation were grown in aeratedliquid cultures. The protonemata were comminuted every 9 days andtransferred into fresh culture medium.

f) Cultivation of Phytophthora infestans

Initially, a cDNA library of Phytophthora infestans was prepared. Tothis end, it is possible to obtain strain ATCC 48886 from the AmericanType Culture Collection Rockville, USA. As a variation of the cultureprotocol described for the strain ATCC 48886, Phytophthora spores werewashed with cold, doubly distilled water and kept in a fridge for 6hours to induce sporulation. The material was then transferred into peamedium. To this end, 150 g of deep-frozen peas (Iglu, obtainable fromlocal supermarkets) were autoclaved under sterile conditions and 1 literof water for 20 minutes. The material was grown in 100-ml-flasks at roomtemperature, on an orbitalshaker (200 rpm). After two days, 2 flaskswere filtered off and the filter residue was comminuted in liquidnitrogen, using mortar and pestle, and for the following 4 days, thisprocedure was repeated for in each case 2 flasks.

Example 2 Isolation of Total DNA from Plants and Microorganisms Such asThraustochytrium and Crypthecodinium for Hybridization Experiments

The details on the isolation of total DNA refer to the work-up of plantmaterial with a fresh weight of one gram.

CTAB buffer: 2% (w/v) N-acetyl-N,N,N-trimethylammonium bromide (CTAB);100 mM Tris-HCl, pH 8.0; 1.4 M NaCl; 20 mM EDTA.

N-Laurylsarcosine buffer: 10% (w/v) N-laurylsarcosine; 100 mM Tris-HCl,pH 8.0; 20 mM EDTA.

The plant material or Crypthecodinium or Thraustochytrium cell materialwas triturated under liquid nitrogen in a mortar to give a fine powderand transferred into 2 ml Eppendorf vessels. The frozen plant materialwas then covered with a layer of 1 ml of decomposition buffer (1 ml CTABbuffer, 100 ml N-laurylsarcosine buffer, 20 ml β-mercaptoethanol and 10ml proteinase K solution, 10 mg/ml) and incubated at 60° C. for one hourwith continuous shaking. The homogenate obtained was distributed intotwo Eppendorf vessels (2 ml) and extracted twice by shaking with anequal volume of chloroform/isoamyl alcohol (24:1). For phase separation,centrifugation was carried out at 8000×g and RT (=room temperature=−23°C.) for 15 minutes in each case. The DNA was then precipitated at −70°C. for 30 minutes using ice-cold isopropanol. The precipitated DNA wassedimented at 4° C. and 10,000 g for 30 minutes and resuspended in 180ml of TE buffer (Sambrook et al., 1989, Cold Spring Harbor LaboratoryPress: ISBN 0-87969-309-6). For further purification, the DNA wastreated with NaCl (1.2 M final concentration) and precipitated again at−70° C. for 30 minutes using twice the volume of absolute ethanol. Aftera wash step with 70% ethanol, the DNA was dried and subsequently takenup in 50 ml of H₂O+RNase (50 mg/ml final concentration). The DNA wasdissolved overnight at 4° C. and the RNase cleavage was subsequentlycarried out for 1 hour at 37° C. The DNA was stored at 4° C.

Example 3 Isolation of Total RNA and Poly(A)+ RNA from Plants andMicroorganisms (Crypthecodinium and Thraustochytrium)

Total RNA was isolated from plants such as linseed and oilseed rape by amethod described by Logemann et al (1987, Anal. Biochem. 163, 21). Thetotal RNA from moss can be obtained from protonema tissue using the GTCmethod (Reski et al., 1994, Mol. Gen. Genet., 244:352-359).

RNA Isolation from Crypthecodinium and Thraustochytrium:

Frozen samples of algae (−70° C.) are triturated in an ice-cold mortarunder liquid nitrogen to give a fine powder. 2 volumes of homogenizationmedium (12.024 g sorbitol, 40.0 ml 1M Tris-HCl, pH 9 (0.2 M); 12.0 ml 5M NaCl (0.3 M), 8.0 ml 250 mM EDTA, 761.0 mg EGTA, 40.0 ml 10% SDS aremade up to 200 ml with H₂O and the pH is brought to 8.5) and 4 volumesof phenol with 0.2% mercaptoethanol are added to the frozen cell powderat 40-50° C. while mixing thoroughly. Then, 2 volumes of chloroform areadded and the mixture is stirred vigorously for 15 minutes. The mixtureis centrifuged for 10 minutes at 10,000 g and the aqueous phase isextracted with phenol/chloroform (2 vol/2 vol) and then with chloroform.

The resulting volume of the aqueous phase is treated with 1/20 volume of4 M sodium acetate (pH 6) and 1 volume of (ice-cold) isopropanol, andthe nucleic acids are precipitated at −20° C. The mixture is centrifugedfor 30 minutes at 10,000 g and the supernatant is removed by suction.This is followed by a wash step with 70% EtOH and another centrifugationstep. The sediment is taken up in Tris borate buffer (80 mM Tris boratebuffer, 10 mM EDTA, pH 7.0). The supernatant is then treated with 1/3vol of 8 M LiCl, mixed are incubated for 30 minutes at 4° C. Afterrecentrifugation, the sediment is washed with 70% ethanol, centrifuged,and the sediment is dissolved in RNase free water.

Poly(A)+-RNA is isolated using Dyna Beads® (Dynal, Oslo, Finland)following the instructions in the manufacturer's protocol.

After the RNA or poly(A)+-RNA concentration has been determined, the RNAis precipitated by adding 1/10 volume 3 M sodium acetate, pH 4.6 and 2volumes of ethanol and stored at −70° C.

For the analysis, 20 μg portions of RNA are separated in aformaldehyde-containing 1.5% strength agarose gel and transferred tonylon membranes (Hybond, Amersham). Specific transcripts are detected asdescribed by Amasino ((1986) Anal. Biochem. 152, 304)).

Isolation of Total RNA and Poly (A)+ RNA from Phytophthora infestans:

Total RNA was obtained using the RNeasy Plant Total RNA kit (Quiagen,Milden) and the buffer contained therein, following the instructions ofthe manufacturer. From the total RNA thus obtained, the poly-(A)+ RNAwas isolated using the Poly Attract in RNA Isolation System III fromPromega (Heidelberg), following the instructions of the manufacturer.

Example 4 Construction of the cDNA Library

To construct the cDNA library from Physcomitrella, Crypthecodinium andThraustochytrium, respectively, the first-strand synthesis was carriedout using murine leukemia virus reverse transcriptase (Roche, Mannheim,Germany) and oligo-d(T) primers, while the second-strand synthesis wascarried out by incubation with DNA polymerase I, Klenow enzyme andcleavage with RNase H at 12° C. (2 hours), 16° C. (1 hour) and 22° C. (1hour). The reaction was quenched by incubation at 65° C. (10 minutes)and subsequently transferred to ice. Double-stranded DNA molecules weremade blunt-ended with T4 DNA polymerase (Roche, Mannheim) at 37° C. (30minutes). The nucleotides were removed by extraction withphenol/chloroform and Sephadex G50 spin columns. EcoRI/XhoI adapters(Pharmacia, Freiburg, Germany) were ligated to the cDNA ends by means ofT4 DNA ligase (Roche, 12° C., overnight), recut with XhoI andphosphorylated by incubation with polynucleotide kinase (Roche, 37° C.,30 minutes). This mixture was subjected to separation on a low-meltingagarose gel. DNA molecules of over 300 base pairs were eluted from thegel, extracted with phenol, concentrated on Elutip D columns (Schleicherand Schüll, Dassel, Germany), ligated to vector arms and packaged intolambda-ZAPII phages or lambda-ZAP-express phages using the Gigapack Goldkit (Stratagene, Amsterdam, the Netherlands), using the manufacturer'smaterial and following their instructions.

The construction of a cDNA library for Phytophthora infestans wascarried out as described above.

Example 5 DNA Sequencing and Computer Analysis

cDNA libraries as described in Example 4 were used for DNA sequencing bystandard methods, in particular the chain termination method using theABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (PerkinElmer, Weiterstadt, Germany). Following the plasmid preparation fromcDNA libraries, individual random clones were sequenced via in-vivo massexcision and retransformation of DH10B on agar plates (details onmaterials and protocol: Stratagene, Amsterdam, the Netherlands). PlasmidDNA was prepared from E. coli cultures grown overnight in Luria brothsupplemented with ampicillin (see Sambrook et al. (1989) (Cold SpringHarbor Laboratory Press: ISBN 0-87969-309-6)) using a Qiagen DNApreparation robot (Qiagen, Hilden) following the manufacturer'sprotocols. Sequencing primers with the following nucleotide sequenceswere used:

5′-CAGGAAACAGCTATGACC-3′ (SEQ ID NO: 13) 5′-CTAAAGGGAACAAAAGCTG-3′(SEQ ID NO: 14) 5′-TGTAAAACGACGGCCAGT-3′ (SEQ ID NI: 15)

The sequences were processed and recorded using the EST-MAX standardsoftware package which is commercially available from Bio Max (Munich,Germany). Exploiting comparative algorithms and using a searchingsequence, homologous genes were searched for using the BLAST program(Altschul et al. (1997) “Gapped BLAST and PSI BLAST: a new generation ofprotein database search programs”, Nucleic Acids Res. 25:3389-3402). Onesequence from Crypthecodinium and Thraustochytrium with homologies withthe search sequence of the Physcomitrella patens moss elongase werecharacterized in greater detail.

Example 6a Identification of the Tc_PSE1 (SEQ ID NO: 3) and Tc_PSE2 (SEQID NO: 5) Gene (Tc=Thraustochytrium) and of the Cc_PSE1 (SEQ ID NO: 7)and Cc_PSE2 Gene (Cc=Crypthecodinium cohnii) by comparison with thePhyscomitrella patens Pp_PSE1 (SEQ ID NO: 1) Gene

The full length sequence of the Pp_PSE1 moss elongase (SEQ ID NO: 2)according to the invention (name: see also Table 2) was employed for thesequence comparisons in the TBLASTN search algorithm:

(SEQ ID NO: 2) MEVVERFYGE LDGKVSQGVN ALLGSFGVEL TDTPTTKGLPLVDSPTPIVL GVSVYLTIVI GGLLWIKARD LKPRASEPFLLQALVLVHNL FCFALSLYMC VGIAYQAITW RYSLWGNAYNPKHKEMAILV YLFYMSKYVE FMDTVIMILK RSTRQISFLHVYHHSSISLI WWAIAHHAPG GEAYWSAALN SGVHVLMYAYYFLAACLRSS PKLKNKYLFW GRYLTQFQMF QFMLNLVQAYYDMKTNAPYP QWLIKILFYY MISLLFLFGN FYVQKYIKPS DGKQKGAKTE.

The complete nucleotide sequence of the moss elongase Pp_PSE1 CDNA iscomposed of approximately 1200 bp. It contains an open reading frame of873 bp which encodes 290 amino acids with a calculated molecular mass of33.4 Da. The protein sequence only has 38.5% identity and 48.3%similarity with a Saccharomyces cerevisiae gene product, for example theSaccharomyces cerevisiae PSE1 gene product, which is required in yeastfor the elongation of fatty acids with medium chain length (Toke &Martin, 1996, Isolation and characterization of a gene affecting fattyacid elongation in Saccharomyces cerevisiae. Journal of BiologicalChemistry 271, 18413-18422).

The EST sequences CC001042041R, TC002034029R and TC002014093R were firstconsidered as target gene amongst other candidate genes owing toinitially weak homologies with the Physcomitrella patens elongase (seeTable 2), the PSE1 gene. FIG. 5 shows the result of the comparison ofthe Pp_PSE1 peptide sequence with the found sequence. It is part of thenucleic acid of Seq ID NO:3 according to the invention (gene name:TcPSE1, inventors' database No. TC002034029R). Letters indicateidentical amino acids, while the plus symbol denotes a chemicallysimilar amino acid. The identities and homologies of all sequences foundin accordance with the invention can be seen from the summary in Table3.

Sequencing of the complete cDNA fragment from clone TC002034029Rresulted in a sequence of 693 base pairs starting with the first base inthe open reading frame. The sequence encodes a polypeptide of 195 aminoacids shown in Seq ID NO:4 with a stop codon in base pair positiontranslated from Seq ID NO:3 in base pair position 586-588. CloneTC002014093R comprises a virtually complete elongase polypeptide as canbe seen from the sequence alignment in FIG. 7. Lines denote identicalamino acids, while colons and dots represent chemically exchangeable,i.e. chemically equivalent, amino acids. The alignment was carried outusing Henikoff & Henikoff's BLOSUM62 amino acid substitution matrix((1992) Amino acid substitution matrices from protein blocks. Proc.Natl. Acad. Sci. USA 89: 10915-10919). Parameters used: Gap Weight: 8;Average Match: 2.912, Length Weight: 2, Average Mismatch: −2.003.

Furthermore, a second EST was identified by the sequence alignment. Thealignment of the Pp_PSE1 peptide sequence with the found sequence isshown in FIG. 6. Even though the homology amongst the parameters chosenis restricted to a few amino acids, this refers to a highly conservedregion of the PUFA specific elongases. The sequence of the completecloned fragment was therefore determined.

Sequencing of the complete cDNA fragment of clone TC002014093R resultedin a sequence of 955 base pairs starting with the first base in the openreading frame. This is referred to by SEQ ID NO:5 according to theinvention. The sequence encodes a polypeptide of 297 amino acids with astop codon in base pair position 892-894 shown in accordance with theinvention in SEQ ID NO: 6.

The Crypthecodinium cohnii EST CC001042041R which encodes the Cc_PSE1gene was identified with the aid of the sequence PpPSE1. The isolatedEST CC001042041R, shown in accordance with the invention as SEQ ID NO:7,is 708 base pairs long and has an open reading frame of 642 base pairsfrom the first base which encodes 214 amino acids and has a stop codonin position 643-645. The amino acid sequence up to the stop codon isshown in accordance with the invention in SEQ ID NO:8.

Besides the similarity with the PSE1 gene product, the similarity withthe Saccharomyces cerevisiae elongase (sce elo 1P), which is required inyeast for the elongation of fatty acids with medium chain length, mayalso be resorted to (Toke & Martin, 1996, Isolation and characterizationof a gene affecting fatty acid elongation in Saccharomyces cerevisiae.Journal of Biological Chemistry 271, 18413-18422). Table 3 shows theidentities and homologies of elongases according to the invention witheach other and with the Physcomitrella patens and yeast elongases. Thedata were obtained with the aid of the GAP program as subprogram of thefollowing software: Wisconsin Package Version 10.0 (Genetics ComputerGroup (GCG), Madison, Wis., USA).

TABLE 3 Identity/ Tc_PSE1 TC_PSE2 Pp_PSE1 homology (SEQ ID NO: 4) (SEQID NO: 6) (SEQ ID NO: 2) Sce elo 1P Cc_PSE1 47.1%/ 50.6%/ 38.5%/ 45.1%/40.2% 43.5% 29.4% 33.5% Tc_PSE1 100/100 n.d. 43.2%/ 41.9%/ 32.7% 29.9%Tc_PSE2 41.7%/ 100/100 39.2%/30.0 35.4%/ 29.5% 27.8%

In particular, FIGS. 5 to 10 can be used to derive the followingsequence motifs as regions of high homology and corresponding consensussequences derived therefrom which, by back translating the amino acidsinto three-base-pair codons, lead to oligonucleotides which can beexploited for isolating novel elongases by means of polymerase chainreaction. They are, in particular, the sequence motifs shown in FIG. 10.These motifs can be used for deriving oligonucleotides which, incombination with two oligonucleotides, can be employed in PCRexperiments for isolating further elongase fragments. To do this, it isexpedient to construct and synthesize one oligonucleotide matching theconventionally defined 5′-3′ strand and a second one with anoligonucleotide matching the 3′-5′ strand downstream. This results in adefinable number of primer combinations, which is limited by permutationof the variants which are possible.

In this context, use may also be made of oligo-dT-primers and variantsthereof, for example by the last base allowing specificity for atranscript pool, such as, for example, oligo dT (12-20) X, where X canbe a G, C or T. Also, a second base oligo dT (12-20) XY can be made useof, where X can be a G, C or A, while the Y can be an A, G, C or T.

The above-defined sequences allow 17- to 20mer oligonucleotides to bederived which can be exploited for isolating gene fragments by varyingthe primer combinations and experimental parameters such as thetemperature program, Mg ion concentration and the like. The resultingfragments can be cloned into suitable vectors and the sequence ofresulting clones can be determined by current methods for identifyingnovel elongases.

Example 6b Isolation of the cDNA Clone from Phytophthora infestans

The cDNA clone designated PI001002014R (SEQ ID NO:11) from Phytophthorainfestans was identified from cDNAs sequenced at random, usinghomologies to PUFA elongase from the moss Physcomitrella patens (ATCCC48886). The clone contains the consensus motif MyxYYF shown in FIG. 8,where, different from PUFA elongases which have hitherto beenidentified, a threonine radical was found as variable amino acid x. Thisfurther variation can be used for deriving PCR primers.

Example 7 Identification of Genes by Means of Hybridization(TC002034029R-11 iGenTc-PCE1)

Gene sequences can be used for identifying homologous or heterologousgenes from cDNA libraries or genomic libraries.

Homologous genes (i.e. full length cDNA clones which are homologous, orhomologs) can be isolated via nucleic acid hybridization using, forexample, cDNA libraries: the method can be made use of in particular forisolating functionally active full length genes of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11. Dependingon the frequency of the gene of interest, 100,000 up to 1,000,000recombinant bacteriophages are plated and transferred to a nylonmembrane. After denaturation with alkali, the DNA is immobilized on themembrane, for example by UV crosslinking. Hybridization is performedunder highly stringent conditions. The hybridization and the wash stepsare carried out in aqueous solution at an ionic strength of 1 M NaCl anda temperature of 68° C. Hybridization probes were generated for exampleby labeling with radioactive (³²P—) nick transcription (High Prime,Roche, Mannheim, Germany). The signals are detected by autoradiography.

Partially homologous or heterologous genes which are related but notidentical can be identified analogously to the process described aboveusing low-stringency hybridization and wash conditions. For the aqueoushybridization, the ionic strength was usually kept at 1 M NaCl, and thetemperature was lowered gradually from 68 to 42° C.

The isolation of gene sequences which only exhibit homologies with anindividual domain of, for example, 10 to 20 amino acids can be carriedout using synthetic, radiolabeled oligonucleotide probes. Radiolabeledoligonucleotides are generated by phosphorylating the 5′ end of twocomplementary oligonucleotides with T4 polynucleotide kinase. Thecomplementary oligonucleotides are hybridized and ligated with eachother to give rise to concatemers. The double stranded concatemers areradiolabeled for example by nick transcription. Hybridization is usuallycarried out under low stringency conditions using high oligonucleotideconcentrations.

Oligonucleotide Hybridization Solution:

6×SSC

0.01 M sodium phosphate

1 mM EDTA (pH 8)

0.5% SDS

100 μg/ml denaturated salmon sperm DNA

0.1% dry low-fat milk

During the hybridization, the temperature is lowered stepwise to 5 to10° C. below the calculated oligonucleotide temperature or to roomtemperature (unless otherwise specified, RT=−23° C. in all experiments),followed by wash steps and autoradiography. Washing is carried out atextremely low stringency, for example 3 wash steps using 4×SSC. Furtherdetails are as described by Sambrook, J., et al. (1989), “MolecularCloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, orAusubel, F. M., et al. (1994) “Current Protocols in Molecular Biology”,John Wiley & Sons.

The clone TC002034029R-11 with the gene name Tc_PCE1_(—)1 is afull-length sequence of an elongase from Thraustochytrium and thuslonger than the clone TC002034029R from Seq. ID No. 3 and Seq. ID No. 4.The clone was isolated using a hybridization method, as described above(Example 7). It is a DNA sequence of a length of 1 050 base pairsencoding for 271 amino acids having a start codon in base pair position43-45 and a stop codon in base pair position 856-858.

Example 8 Identification of Target Genes by Screening ExpressionLibraries with Antibodies

To generate recombinant protein, for example in E. coli, cDNA sequencesare used (for example Qiagen QIAexpress pQE system). The recombinantproteins are then affinity-purified, usually via Ni-NTA affinitychromatography (Qiagen). The recombinant proteins are then used forraising specific antibodies, for example using standard techniques forimmunizing rabbits. The antibodies are then affinity purified using anNi NTA column which is presaturated with recombinant antigen, asdescribed by Gu et al., (1994) BioTechniques 17:257 262. The antibodycan then be used for screening expression cDNA libraries byimmunological screening (Sambrook, J., et al. (1989), “MolecularCloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, orAusubel, F. M., et al. (1994) “Current Protocols in Molecular Biology”,John Wiley & Sons).

Example 9 Plasmids for Plant Transformation

Binary vectors such as pBinAR can be used for plant transformation(Höfgen and Willmitzer, Plant Science 66 (1990) 221-230). The binaryvectors can be constructed by ligating the cDNA in sense or antisenseorientation into T DNA. 5′ of the cDNA, a plant promoter activates cDNAtranscription. A polyadenylation sequence is located 3′ of the cDNA.

Tissue specific expression can be achieved using a tissue specificpromoter. For example, seed-specific expression can be achieved bycloning in the napin or the LeB4 or the USP promoter 5′ of the cDNA.

Any other seed specific promoter element can also be used. The CaMV-35Spromoter may be used for constitutive expression in all of the plant.

The protein expressed can be targeted into a cellular compartment usinga signal peptide, for example for plastids, mitochondria or theendoplasmatic reticulum (Kermode, Crit. Rev. Plant Sci. 15, 4 (1996)285-423). The signal peptide is cloned 5′ in correct reading frame withthe cDNA in order to achieve subcellular localization of the fusionprotein.

Example 10 Transformation of Agrobacterium

Agrobacterium mediated plant transformation can be carried out forexample using the Agrobacterium tumefaciens strain GV3101 (pMP90) (Konczand Schell, Mol. Gen. Genet. 204 (1986) 383-396) or LBA4404 (Clontech).The transformation can be carried out by standard transformationtechniques (Deblaere et al., Nucl. Acids. Tes. 13 (1984), 4777-4788).

Example 11 Plant Transformation

Agrobacterium mediated plant transformation can be carried out usingstandard transformation and regeneration techniques (Gelvin, Stilton B.,Schilperoort, Robert A., Plant Molecular Biology Manual, 2nd Ed.,Dordrecht: Kluwer Academic Publ., 1995, in Sect., Ringbuc ZentraleSignatur: BT11 P ISBN 0-7923-2731-4; Glick, Bernard R., Thompson, JohnE., Methods in Plant Molecular Biology and Biotechnology, Boca Raton:CRC Press, 1993, 360 pp. ISBN 0-8493-5164-2).

For example, oilseed rape can be transformed by means of cotyledon orhypocotyledon transformation (Moloney et al., Plant Cell Report 8 (1989)238 242; De Block et al., Plant Physiol. 91 (1989) 694-701). The use ofantibiotics for the selection of agrobacteria and plants depends on thebinary vector and the agrobacterial strain used for the transformation.The selection of oilseed rape is normally carried out using kanamycin asselectable plant marker.

Agrobacterium mediated gene transfer in linseed (Linum usitatissimum)can be carried out for example using a technique described by Mlynarovaet al. (1994) Plant Cell Report 13:282-285.

The transformation of soya can be carried out for example using atechnique described in EP-A-0 424 047 (Pioneer Hi-Bred International) orin EP-A-0 397 687, U.S. Pat. No. 5,376,543, U.S. Pat. No. 5,169,770(University of Toledo).

Plant transformation using particle bombardment,polyethylene-glycol-mediated DNA uptake or via the silicon carbonatefiber technique is described, for example, by Freeling and Walbot “Themaize handbook” (1993) ISBN 3-540-97826-7, Springer Verlag New York).

Example 12 Plasmids for Plant Transformation

Binary vectors such as pBinAR can be used for plant transformation(Höfgen and Willmitzer, Plant Science 66 (1990) 221-230). The binaryvectors can be constructed by ligating the cDNA in sense or antisenseorientation into T-DNA. 5′ of the cDNA, a plant promoter activates cDNAtranscription. A polyadenylation sequence is located 3′ of the cDNA.Tissue specific expression can be achieved using a tissue specificpromoter. For example, seed specific expression can be achieved bycloning in the napin or the LeB4 or the USP promoter 5′ of the cDNA. Anyother seed specific promoter element can also be used. The CaMV-35Spromoter may be used for constitutive expression in all of the plants.In particular, genes encoding elongases and desaturases can be clonedinto a binary vector by constructing a plurality of expression cassettesin succession in order to imitate the metabolic pathway in the plant.

Within an expression cassette, the protein expressed can be targetedinto a cellular compartment using a signal peptide, for example forplastids, mitochondria or the endoplasmatic reticulum (Kermode, Crit.Rev. Plant Sci. 15, 4 (1996) 285-423). The signal peptide is cloned 5′in correct reading frame with the cDNA in order to achieve subcellularlocalization of the fusion protein.

Example 13 In Vivo Mutagenesis

The in vivo mutagenesis of microorganisms can be performed by passagingthe plasmid DNA (or any other vector DNA) via E. coli or othermicroorganisms (for example Bacillus spp. or yeasts such asSaccharomyces cerevisiae), in which the ability of retaining theintegrity of their genetic information is disrupted. Conventionalmutator strains have mutations in the genes for the DNA repair system(for example mutHLS, mutD, mutT and the like; as reference, see Rupp, W.D. (1996) DNA repair mechanisms, in: Escherichia coli and Salmonella,pp. 2277-2294, ASM: Washington). These strains are known to the skilledworker. The use of these strains is illustrated for example in Greener,A., and Callahan, M. (1994) Strategies 7:32-34. Mutated DNA moleculesare preferably transferred to plants after the microorganisms have beenselected and tested. Transgenic plants are generated in accordance withvarious examples in the examples section of the present document.

Example 14 Studying the Expression of a Recombinant Gene Product in aTransformed Organism

The activity of a recombinant gene product in the transformed hostorganism was measured at the transcriptional and/or the translationallevel.

A suitable method for determining the amount of transcription of thegene (which indicates the amount of RNA available for translation of thegene product) is to carry out a northern blot as specified hereinbelow(for reference, see Ausubel et al. (1988) Current Protocols in MolecularBiology, Wiley: New York, or the abovementioned examples section) inwhich a primer which is designed such that it binds to the gene ofinterest is labeled with a detectable label (usually radioactivity orchemiluminescence) so that, when the total RNA of a culture of theorganism is extracted, separated on a gel, transferred to a stablematrix and incubated with this probe, binding and the extent of bindingof the probe indicates the presence as well as the quantity of the mRNAfor this gene. This information indicates the degree of transcription ofthe transformed gene. Total cell RNA can be prepared from cells, tissuesor organs by a plurality of methods, all of which are known in the art,such as, for example, the method of Bormann, E. R., et al. (1992) Mol.Microbiol. 6:317-326.

Northern Hybridization:

For the RNA hybridization, 20 μg of total RNA or 1 μg of poly(A)⁺-RNAwere separated by gel electrophoresis in 1.25% strength agarose gelsusing formaldehyde as described by Amasino (1986, Anal. Biochem. 152,304), transferred to positively charged nylon membranes (Hybond N+,Amersham, Brunswick) by capillary attraction using 10×SSC, immobilizedby UV light and prehybridized for 3 hours at 68° C. using hybridizationbuffer (10% dextran sulfate w/v, 1 M NaCl, 1% SDS, 100 mg herring spermDNA). The DNA probe had been labeled with the Highprime DNA labeling kit(Roche, Mannheim, Germany) during the prehybridization stage usingα-³²P-dCTP (Amersham, Brunswick, Germany). The hybridization was carriedout after adding the labeled DNA probe in the same buffer at 68° C.overnight. The wash steps were carried out twice for 15 minutes using2×SSC and twice for 30 minutes using 1×SSC, 1% SDS, at 68° C. The sealedfilters were exposed at −70° C. for a period of 1 to 14 days.

Standard techniques, such as a Western blot (see, for example, Ausubelet al. (1988) Current Protocols in Molecular Biology, Wiley: New York)can be employed for studying the presence or the relative quantity ofprotein translated by this mRNA. In this method, the total cell proteinsare extracted, separated by means of gel electrophoresis, transferred toa matrix such as nitrocellulose and incubated with a probe such as anantibody which specifically binds to the desired protein. This probe isusually provided with a chemiluminescent or colorimetric label which canbe detected readily. The presence and the quantity of the label observedindicates the presence and the quantity of the desired mutated proteinwhich is present in the cell.

Example 15 Analysis of the Effect of the Recombinant Proteins on theProduction of the Desired Product

The effect of the genetic modification in plants, fungi, algae, ciliatesor on the production of a desired compound (such as a fatty acid) can bedetermined by growing the modified microorganisms or the modified plantunder suitable conditions (such as those described above) and analyzingthe medium and/or the cell components for the increased production ofthe desired product (i.e. of lipids or a fatty acid). These analyticaltechniques are known to the skilled worker and encompass spectroscopy,thin layer chromatography, various staining methods, enzymatic andmicrobiological methods, and analytical chromatography such as highperformance liquid chromatography (see, for example, Ullman,Encyclopedia of Industrial Chemistry, Vol. A2, pp. 89-90 and pp.443-613, VCH Weinheim (1985); Fallon, A., et al., (1987) “Applicationsof HPLC in Biochemistry” in: Laboratory Techniques in Biochemistry andMolecular Biology, Vol. 17; Rehm et al. (1993) Biotechnology, Vol. 3,Chapter III “Product recovery and purification”, pp. 469-714, VCHWeinheim; Belter, P. A., et al. (1988) Bioseparations: downstreamprocessing for Biotechnology, John Wiley and Sons; Kennedy, J. F., andCabral, J. M. S. (1992) Recovery processes for biological Materials,John Wiley and Sons; Shaeiwitz, J.A., and Henry, J. D. (1988)Biochemical Separations, in: Ullmann's Encyclopedia of IndustrialChemistry, Bd. B3; Chapter 11, Vol. 1-27, VCH Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, NoyesPublications).

In addition to the abovementioned methods, plant lipids are extractedfrom plant material as described by Cahoon et al. (1999) Proc. Natl.Acad. Sci. USA 96 (22):12935-12940, and Browse et al. (1986) AnalyticBiochemistry 152:141-145. Qualitative and quantitative lipid or fattyacid analysis is described in Christie, William W., Advances in LipidMethodology, Ayr/Scotland: Oily Press (Oily Press Lipid Library; 2);Christie, William W., Gas Chromatography and Lipids. A PracticalGuide—Ayr, Scotland: Oily Press, 1989, Repr. 1992, IX, 307 pp. (OilyPress. Lipid Library; 1); “Progress in Lipid Research, Oxford: PergamonPress, 1 (1952)-16 (1977) under the title: Progress in the Chemistry ofFats and Other Lipids CODEN.

In addition to measuring the end product of the fermentation, it is alsopossible to analyze other components of the metabolic pathways which areused for producing the desired compound, such as intermediates andbyproducts, in order to determine the overall production efficiency ofthe compound. The analytical methods encompass measurements of thenutrient quantities in the medium (for example sugars, carbohydrates,nitrogen sources, phosphate and other ions), biomass composition andgrowth measurements, analysis of the production of customary metabolitesof biosynthetic pathways, and measurements of gases which are generatedduring fermentation. Standard methods for these measurements aredescribed in Applied Microbial Physiology; A Practical Approach, P. M.Rhodes and P. F. Stanbury, Ed., IRL Press, pp. 131-163 and 165-192(ISBN: 0199635773) and references stated therein.

One example is the analysis of fatty acids (abbreviations: FAME, fattyacid methyl ester; GC-MS, gas liquid chromatography/mass spectrometry;TAG, triacylglycerol; TLC, thin layer chromatography).

The unambiguous detection of the presence of fatty acid products can beobtained by analyzing recombinant organisms by analytical standardmethods: GC, GC-MS or TLC, as they are described on several occasions byChristie and the references therein (1997, in: Advances on LipidMethodology, Fourth edition: Christie, Oily Press, Dundee, 119-169;1998, gas chromatography/mass spectrometry methods, Lipide 33:343-353).

The material to be analyzed can be disrupted by sonication, grinding ina glass mill, liquid nitrogen and grinding or via other applicablemethods. After disruption, the material must be centrifuged. Thesediment is resuspended in distilled water, heated for 10 minutes at100° C., ice cooled and recentrifuged followed by extraction in 0.5 Msulfuric acid in methanol with 2% dimethoxypropane for 1 hour at 90° C.,which leads to hydrolyzed oil and lipid compounds which givetransmethylated lipids. These fatty acid methyl esters are extracted inpetroleum ether and finally subjected to GC analysis using a capillarycolumn (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 μm, 0.32 mm) at atemperature gradient of between 170° C. and 240° C. for 20 minutes and 5minutes at 240° C. The identity of the resulting fatty acid methylesters must be defined using standards which are commercially available(i.e. Sigma).

In the case of fatty acids for which no standards are available, theidentity must be demonstrated via derivatization followed by GC/MSanalysis. For example, the localization of fatty acids with triple bondmust be demonstrated via GC/MS following derivitization with4,4-dimethoxyoxazolin derivatives (Christie, 1998, see above).

Example 16 Expression Constructs in Heterologous Microbial Systems

Strains, Growth Conditions and Plasmids

The Escherichia coli strain XL1 Blue MRF′ kan (Stratagene) is used forsubcloning the novel Physcomitrella patens elongases, such as PpPSE1.For functionally expressing this gene, we used the Saccharomycescerevisiae strain INVSc 1 (Invitrogen Co.). E. coli is cultured at 37°C. in Luria Bertini broth (LB, Duchefa, Haarlem, the Netherlands). Ifnecessary, ampicillin (100 mg/liter) is added, and 1.5% of agar (w/v) isadded for solid LB media. S. cerevisiae is cultured at 30° C. either inYPG medium or in complete minimal medium without uracil (CMdum; see:Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J.G., Smith, J. A., Struhl, K., Albright, L. B., Coen, D. M., and Varki,A. (1995) Current Protocols in Molecular Biology, John Wiley & Sons, NewYork) together with 2% (w/v) of either raffinose or glucose. For solidmedia, 2% (w/v) of Bacto™ agar (Difco) are added. The plasmids used forcloning and expression are pUC18 (Pharmacia) and pYES2 (Invitrogen Co.).

Example 17 Cloning and Expression of PUFA Specific Physcomitrella,Crypthecodinium and Thraustochytrium elongases

The full length genes of sequences according to the invention can beisolated as described in Example 7 and processed as illustratedhereinbelow. Concrete expression examples are shown with regard to theuse.

a) Elongation of Fatty Acids by the Moss Elongase Pp_PSE1:

For expression in yeast, the P. patens cDNA clone PpPSE1 (earlierdatabase sequence name: 08_ck19_b07, new name: pp001019019f), whichencodes the PUFA specific elongase (PSE1) gene, was first modified insuch a way that a BamHI restriction site and the yeast consensussequence for highly effective translation (Kozak, M. (1986) Pointmutations define a sequence flanking the AUG initiator codon thatmodulates translation by eukaryotic ribosomes, Cell 44, 283-292) wasobtained next to the start codon and a BamHI restriction site wasobtained which flanked the stop codon. To amplify the open readingframe, a primer pair which was complementary to its 5′ and 3′ ends wassynthesized.

The gene of the sequence according to the invention shown in SEQ ID NO:1was cloned into pYES by means of polymerase chain reaction, giving riseto the plasmid pYPp_PSE1:

The following oligonucleotides were employed for the PCR experiment:

Ppex6: ggatccacataatggaggtcgtggagag (SEQ ID NO: 16) attc Ppex6r:ggatcctcactcagttttagctcctttt (SEQ ID NO: 17) gc

The PCR reaction was carried out with plasmid DNA of clone PP001019019Fas matrix in a thermocycler (Biometra) with Pfu DNA (Stratagene)polymerase and the following temperature program: 3 minutes at 96° C.followed by 25 cycles with 30 seconds at 96° C., 30 seconds at 55° C.and 1 minute at 72° C., 1 cycle with 10 minutes at 72° C.

The correct size of the amplified DNA fragment was confirmed by agaroseTBE gel electrophoresis. The amplified DNA was extracted from the gelusing the QIAquick gel extraction kit (QIAGEN) and initially cloned intopUC18 using the Sure Clone Ligation Kit (Pharmacia). The fragment clonedthus was cut with BamHI and ligated into pYES, giving rise to pYPp_PSE1.The fragment orientation was checked by means of HindIII. Following thetransformation of E. coli XL1 Blue MRF′ kan, a DNA minipreparation(Riggs, M. G., & McLachlan, A. (1986) A simplified screening procedurefor large numbers of plasmid mini preparation. BioTechniques 4, 310-313)of transformants was carried out, and positive clones were identified bymeans of BamHI restriction analysis. The sequence of the cloned PCRproduct was confirmed by resequencing using the ABI PRISM Big DyeTerminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer,Weiterstadt).

One clone was grown with the Nucleobond® AX 500 plasmid DNA extractionkit (Macherey Nagel, Düringen) for the DNA maxipreparation.

Saccharomyces INVSc1 was transformed with pYPp_PSE1 or with pYES2 ascontrol by means of a modified PEG/lithium acetate protocol (Ausubel etal., 1995). Following selection on CMdum agar plates with 2% glucose,transformants and a pYES2 transformant were selected for furthercultivation and functional expression as already stated and fed variousfatty acids in the medium.

-   i) Lipid patterns of yeasts which are transformed with the pYES    plasmid without fragment insertion or which express the Pp-PSE1 gene    (data in mol %) after feeding with 250 μm hexadecatrienoic acid    (16:3^(Δ) ^(7c,10c,13c) ).

TABLE 4 PYES2 PYES2 pYPp_PSE1 PYPp_PSE1 16:0 11.8% 16:0 11.1% 16:1 28.7%16:1 23.9% 16:3^(Δ7c,10c,13c) 9.2% 16:3^(Δ7c,10c,13c) 12.0% 18:0 10.6%18:0 8.6% 18:1^(Δ9c) 34.9% 18:1^(Δ9c) 20.6% 18:1^(Δ11c) 1.1% 18:1^(Δ11c)1.4% 18:3^(Δ9c,12c,15c) 3.7% 18:3^(Δ9c,12c,15c) 21.4%

-   ii) Lipid patterns of yeasts which are transformed with the pYES    plasmid without fragment insertion or which express the Pp-PSE1 gene    (data in mol %) after feeding with 500 μm pinolenic acid (18:3^(Δ)    ^(6c,9c,12c) ).

TABLE 5 PYES2 PYES2 pYPp_PSE1 PYPp_PSE1 16:0 18.3% 16:0 16.9% 16:1^(Δ9c)16.0% 16:1^(Δ9c) 15.3% 18:0  8.6% 18:0 8.4% 18:1^(Δ9c) 16.7% 18:1^(Δ9c)17.5% 18:1^(Δ11c)  0.7% 18:1^(Δ11c) 2.0% 18:3^(Δ5c,9c,12c) 39.8%18:3^(Δ5c,9c,12c) 32.6% 20:3^(Δ7c,11c,14c)   0% 20:3^(Δ7c,11c,14c) 5.1%

-   iii) Lipid patterns of yeasts which are transformed with the pYES    plasmid without fragment insertion or which express the Pp-PSE1 gene    (data in mol %) after feeding with 500 μm stearidonic acid (18:4^(Δ)    ^(6c,9c,12c,15c) ).

TABLE 6 PYES2 pYES2 pYPp_PSE1 PYPp_PSE1 16:0 15.2% 16:0 15.6% 16:1^(Δ9c)13.1% 16:1^(Δ9c) 14.9% 18:0 12.3% 18:0 10.7% 18:1^(Δ9c) 12.9% 18:1^(Δ9c)14.0% 18:1^(Δ11c) 0.7% 18:1^(Δ11c) 1.2% 18:3^(Δ6c,9c,12c,15c) 45.4%18:3^(Δ6c,9c,12c,15c) 23.8% 20:4^(Δ8c,11c,14c,17c) 0.4%20:4^(Δ8c,11c,14c,17c) 19.8%

-   iv) Lipid patterns of yeasts which are transformed with the pYES    plasmid without fragment insertion or which express the Pp-PSE1 gene    (data in mol %) after feeding with 500 μm linoleic acid (18:2^(Δ)    ^(9c,12c) ).

TABLE 7 pYES2 pYES2 pYPp_PSE1 PYPp_PSE1 16:0 7.9% 16:0 8.7% 16:1^(Δ9c)1.2% 16:1^(Δ9c) 1.3% 18:0 5.3% 18:0 5.1% 18:1^(Δ9c) 1.3% 18:1^(Δ9c) 1.3%18:2^(Δ9c,12c) 83.9% 18:2^(Δ9c,12c) 80.4% 20:2^(Δ11c,14c) 0.5%20:2^(Δ11c,14c) 3.2%B) Elongation of Fatty Acids by a Thraustochytrium elongase

For expression in yeast, the Thraustochytrium cDNA clone of SEQ ID NO: 3(Tc_PSE2), which encodes a PUFA-specific elongase (PSE) gene, is firstmodified in such a way that it constitutes a functionally activepolypeptide. To this end, the N-terminus of the protein is elongated atDNA level by 42 base pairs by the few missing bases from thePhyscomitrella patens elongase. However, it is also possible only to adda start codon in the correct reading frame for the sequence.

The following oligonucleotides are employed for the PCR experiment:

pTCPSE2-5′: aaaggatccacataatggaggtcgtggagagattctacggtgagttggatgggaagGTCATTTCGGGCCTCGACC (SEQ ID NO: 18) pTCPSE2-3′:aaggatccctgagttttagctcccttttgctttcc (SEQ ID NO: 19)

In addition, both oligonucleotides contain a BamHI restriction site andthe yeast consensus sequence for highly efficient translation (Kozak, M.(1986) Point mutations define a sequence flanking the AUG initiatorcodon that modulates translation by eukaryotic ribosomes, Cell 44,283-292).

The PCR reaction is carried out with plasmid DNA as matrix in athermocycler (Biometra) with Pfu DNA (Stratagene) polymerase and thefollowing temperature program: 3 minutes at 96° C. followed by 25 cycleswith 30 seconds at 96° C., 30 seconds at 55° C. and 3 minutes at 72° C.,1 cycle with 10 minutes at 72° C. and stop at 4° C.

The correct size of the amplified DNA fragment is confirmed by agaroseTBE gel electrophoresis. The amplified DNA is extracted from the gelusing the QIAquick gel extraction kit (QIAGEN) and ligated into theSinai restriction site of the dephosphorylated vector pUC18 using theSure Clone Ligation Kit (Pharmacia), giving rise to pUC-hybrid-Tc_PSE2.Following the transformation of E. coli XL1 Blue MRF′ kan, a DNAminipreparation (Riggs, M. G., & McLachlan, A. (1986) A simplifiedscreening procedure for large numbers of plasmid mini-preparation.BioTechniques 4, 310-313) of 24 ampicillin-resistant transformants wascarried out, and positive clones were identified by means of BamHIrestriction analysis. The sequence of the cloned PCR product wasconfirmed by resequencing using the ABI PRISM Big Dye Terminator CycleSequencing Ready Reaction Kit (Perkin-Elmer, Weiterstadt).

The plasmid DNA of pUC PSE1 and pUC-hybrid-Tc_PSE2 was first cleavedwith BamHI and the fragments obtained were ligated into the BamHIrestriction site of the dephosphorylated yeast/E. coli shuttle vectorpYES2, giving rise to pY2 hybrid-Tc_PSE2. Following transformation of E.coli and DNA minipreparation from the transformants, the orientation ofthe DNA fragment in the vector was checked by cleavage with HindIII. Oneclone was grown with the Nucleobond® AX 500 plasmid DNA extraction kit(Macherey-Nagel, Düringen) for the DNA maxipreparation. SaccharomycesINVSc1 is transformed with pY2PSE1, pYES2, pY2-hybrid-Tc_PSE2 and pYES2by means of a modified PEG/lithium acetate protocol (Ausubel et al.,1995). Following selection on CMdum agar plates with 2% glucose, in eachcase four pY2PSE1 transformants (pY2PSE1a-d), pY2-hybrid-Tc_PSE2transformants (pY2-hybrid-Tc_PSE2 1a-d) and one pYES2 transformant areselected for further culture and functional expression.

Functional Expression of an Elongase Activity in Yeast

Preculture:

20 ml of CMdum liquid medium with 2% (w/v) raffinose were inoculatedwith the transgenic yeast clones (pY2-hybrid-Tc_PSE2 1a-d, pYES2) andcultured for 3 days at 30° C., 200 rpm, until an optical density at 600nm (OD₆₀₀) of 1.5-2 had been reached.

Main Culture:

For expression, 20 ml of CMdum liquid medium with 2% raffinose and 1%(v/v) Tergitol NP 40 were supplemented with fatty acid substrates to afinal concentration of 0.003% (w/v). The media are inoculated with theprecultures to an OD₆₀₀ of 0.05. Expression was induced for 16 hours atan OD₆₀₀ of 0.2, using 2% (w/v) galactose, whereupon the cultures wereharvested at an OD₆₀₀ of 0.8-1.2.

Fatty Acid Analysis

The overall fatty acids were extracted from yeast cultures and analyzedby means of gas chromatography. To this end, cells of 5 ml culture wereharvested by centrifugation (1000×g, 10 minutes, 4° C.) and washed oncewith 100 mM NaHCO₃, pH 8.0, to remove residual medium and fatty acids.To prepare the fatty acid methyl esters (FAMEs), the cell sediments weretreated for 1 hour at 80° C. with 1 M methanolic H₂SO₄ and 2% (v/v)dimethoxypropane. The FAMEs were extracted twice with 2 ml of petroleumether, washed once with 100 mM NaHCO₃, pH 8.0, and once with distilledwater, and dried with Na₂SO₄. The organic solvent was evaporated under astream of argon, and the FAMEs were dissolved in 50 μl petroleum ether.The samples were separated on a ZEBRON ZB Wax capillary column (30 m,0.32 mm, 0.25 μm; Phenomenex) in a Hewlett Packard 6850 gaschromatograph equipped with a flame ionization detector. The oventemperature was programmed from 70° C. (hold for 1 minute) to 200° C. ata rate of 20° C./minute, then to 250° C. (hold for 5 minutes) at a rateof 5° C./minute and finally to 260° C. at a rate of 5° C./minute.Nitrogen was used as the carrier gas (4.5 ml/minute at 70° C.). Thefatty acids were identified by comparison with retention times of FAMEstandards (SIGMA).

The fatty acid patterns of five transgenic yeast strains are shown inTable 1 in mol %.

The ratios of the γ-linolenic acid which had been added and taken up areemphasized by numbers printed in bold, those of the elongated productsby numbers in red and those of the elongated γ-linolenic acid by numbersprinted in bold (last line).

The GC analysis of FAMEs which from total lipids of the yeaststransformed with pYES2 (i/control) and pY2PSE1 (ii-iv c+d/ in each casetransformed with pY2PSE1A, pY2PSE1B, pY2PSE1C, pY2PSE1D) is shown inFIG. 2 a-e. For the analysis, the transgenic yeasts were cultured in thepresence of γ-18:3. Table 1 shows their fatty acid patterns in mol %.The uptake of γ-18:3 is emphasized by numbers printed in bold, theelongation product dihomo-γ-linolenic acid (20:3Δ8,11,14) is underlinedand the ratio γ-18:3 elongation product (also in mol %) is emphasized bynumbers printed in bold (last line). The structure and the mass spectraof the DMOX derivative of cis-Δ6,9,12 C18:3 can be seen from FIG. 3 a+b.The structure and the mass spectra of the DMOX derivative of Δ8,11,14C20:3 can be seen from FIG. 4 a+b.

The results demonstrate that γ-18:3 has been incorporated into alltransgenic yeasts in large amounts. All four transgenic yeast cloneswhich had been transformed with pY2PSE1 exhibit an additional peak inthe gas chromatogram, which was identified as 20:3 Δ8,11,14 by acomparison of the retention times. A gas chromatography/massspectroscopy can provide additional proof to confirm this identity. Thepercentage of elongated γ-18:3 was 23.7 to 40.5%, as shown in Table 1.Furthermore, no significant elongation of palmitic acid (16:0),palmitoleic acid (16:1), stearic acid (18:0) or oleic acid (18:1 Δ9) wasobserved.

The products identified demonstrate that the nucleotide sequence ofPpPSE1 encodes a Δ6-selective fatty acid elongase from the mossPhyscomitrella patens, which leads to the formation of novel fatty acidsin transgenic yeasts.

The ratios of the fatty acid substrates which have been added and takenup can be determined as above, so that quantity and quality of theelongase reaction can be detected.

The structure and the mass spectra of DMOX derivatives also reveal therespective position of a double bond.

Further feeding experiments with a wide range of other fatty acids (forexample arachidonic acid, eicosapentaenoic acid and the like) can becarried out for confirming the substrate selectivity of this elongase ingreater detail.

Example 18 Isolation of the Desired Product from Transformed orOrganisms in General

The desired product can be obtained from plant material or fungi, algae,ciliates, animal cells or from the supernatant of the above describedcultures by various methods known in the art. If the desired product isnot secreted from the cells, the cells can be harvested from the cultureby slow centrifugation, and the cells can be lyzed by standardtechniques, such as mechanical force or sonication. Plant organs can beseparated mechanically from other tissue or other organs. Afterhomogenization, the cell debris is removed by centrifugation, and thesupernatant fraction, which contains the soluble proteins, is retainedfor further isolating the desired compound. If the product is secretedfrom desired cells, the cells are removed from the culture by slowcentrifugation, and the supernatant fraction is retained for the furtherisolation.

The supernatant fraction from each isolation step is subjected to achromatography with a suitable resin, the desired molecule either beingretained on the chromatography resin while many contaminants in thesample are not, or the contaminants remaining on the resin while thesample does not. These chromatography steps can be repeated, if desired,using either the same or other chromatography resins. The skilled workeris familiar with selecting suitable chromatography resins and with theirmost effective use for a particular molecule to be isolated. The productisolated can be concentrated by filtration or ultrafiltration and storedat a temperature at which the stability of the product is highest.

A broad spectrum of isolation methods is known in the art, and theisolation method above is not intended to be limiting. These isolationmethods are described, for example, in Bailey, J. E., & Ollis, D. F.,Biochemical Engineering Fundamentals, McGraw Hill: New York (1986).

Identity and purity of the compounds isolated can be determined bystandard techniques of the art. They include high performance liquidchromatography (HPLC), spectroscopic methods, staining methods, thinlayer chromatography, NIRS, enzyme assays or microbiological methods.For a review of these analytical methods, see: Patek et al. (1994) Appl.Environ. Microbiol. 60:133-140; Malakhova et al. (1996) Biotekhnologiya11:27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19:67-70.Ulmann's Encyclopedia of Industrial Chemistry (1996) Vol. A27, VCHWeinheim, pp. 89-90, pp. 521-540, pp. 540-547, pp. 559-566, 575-581 andpp. 581-587; Michal, G (1999) Biochemical Pathways: An Atlas ofBiochemistry and Molecular Biology, John Wiley and Sons; Fallon, A., etal. (1987) Applications of HPLC in Biochemistry in: LaboratoryTechniques in Biochemistry and Molecular Biology, Vol. 17.

EQUIVALENTS

The skilled worker knows, or can identify, a number of equivalents ofthe specific embodiments according to the invention which have beendescribed herein by simply resorting to routine experiments. Theseequivalents are intended to be covered by the patent claims.

We claim:
 1. A gene construct having a heterologous promoter or a vectorcomprising an isolated nucleic acid sequence encoding a polypeptidefunctionally linked to one or more regulatory signals, wherein theisolated nucleic acid sequence comprises a derivative of SEQ ID NO: 1,and the polypeptide elongates C₁₈-fatty acids with at least two doublebonds in the fatty acid molecule, and wherein the polypeptide isselected from the group consisting of i) polypeptides having the aminoacid sequence of SEQ ID NO:2, and ii) polypeptides having an amino acidsequence with at least 80% identity at the amino acid level with SEQ IDNO:2 having elongase activity.
 2. The gene construct or vector of claim1, wherein the isolated nucleic acid sequence is derived fromPhyscomitrella.
 3. The gene construct or vector as claimed in claim 1,further comprising a nucleic acid sequence which encodes a fatty acidbiosynthesis gene.
 4. The gene construct or vector as claimed in claim2, wherein the nucleic acid which encodes a fatty acid biosynthesis geneis selected from the group consisting of: Δ19-, Δ17-, Δ15-, Δ12-, Δ9-,Δ8-, Δ6-, Δ5-, Δ4-desaturase, hydroxylases, Δ12-acetylenase, acyl-ACPthioesterases, β-ketoacyl-ACP synthases and β-ketoacyl-ACP reductases.5. An organism other than a human comprising the isolated nucleic acidsequence introduced by the gene construct or vector as claimed inclaim
 1. 6. The organism as claimed in claim 5, wherein the organism isa microorganism, yeast or a plant.
 7. The organism as claimed in claim5, wherein the organism is a transgenic plant.
 8. A process for thepreparation of a PUFA, which comprises culturing an organism comprisingthe isolated nucleic acid sequence introduced by the gene construct orvector of claim 1 under conditions under which PUFAs are formed in theorganism.
 9. The process as claimed in claim 8, wherein the PUFA is aC₁₈-fatty acid molecule with at least two double bonds in the fatty acidmolecule.
 10. The process as claimed in claim 9, wherein the C₁₈-fattyacid molecule is isolated from the organism in the form of an oil, lipidor a free fatty acid.
 11. The process as claimed in claim 8, wherein theorganism is a microorganism, an animal or a plant.
 12. The process asclaimed in claim 8, wherein the organism is a transgenic plant.
 13. Theprocess as claimed in claim 8, wherein the C₁₈-fatty acid is a fattyacid with three double bonds in the molecule.
 14. A kit comprising thegene construct or vector comprising said isolated nucleic acid sequenceof claim
 1. 15. The gene construct or vector as claimed in claim 1wherein the isolated nucleic acid sequence is derived from a plant. 16.A gene construct of claim 1, wherein gene expression is enhanced by saidregulatory signals.
 17. The gene construct or vector of claim 1, whereinthe polypeptide having at least 80% identity at the amino acid levelwith SEQ ID NO:2 contains motifs FLHVYHH, corresponding to amino acids158 to 164 of SEQ ID NO: 2 which is optionally substituted in the first,third, fourth and fifth positions position with T, Q, W, L respectively,LMYAYYF, corresponding to amino acids 196 to 202 of SEQ ID NO: 2 whichis optionally substituted in the first, fourth, fifth, and sixth,position with I, M, H, F respectively, and FGNFYVQ, corresponding toamino acids 268 to 274 of SEQ ID NO: 2 which is optionally substitutedin the second and seventh position with L.
 18. The gene construct orvector of claim 1, wherein the polypeptide is selected from the groupsconsisting of i) polypeptides having the amino acid sequence of SEQ IDNO:2, and ii) polypeptides having an amino acid sequence with at least95% identity at the amino acid level with SEQ ID NO:2 having elongaseactivity.
 19. The gene construct or vector of claim 1, wherein thepolypeptide elongates 6,9-octadecadienoic acid, linoleic acid, linolenicacid, α- or γ-linolenic acid, pinolenic acid or stearidonic acid.